Characterization of CC-chemokine receptor 7 expression on murine T cells in lymphoid tissues

Expression of the lymph node homing and CC‐chemokine receptor 7 (CCR7), with L‐selectin (CD62L), has been shown to divide human memory T cells into two functionally distinct subsets. We generated a polyclonal antibody against murine CCR7 and used this antibody to study CCR7 expression on murine T‐cell subsets. Using flow cytometric staining of T cells for visualisation expression of CCR7 in association with CD62L and CD44, a major population of CD4 or CD8 T cells expressing CCR7 were found to be CD62L^high CD44^low, which would suggest a naïve cell phenotype. By analogy with human studies, memory cells could be subdivided into CCR7^high CD62L^high CD44^high (central memory) and CCR7^low CD62L^low CD44^high (effector memory). The proportions of these populations were different in lymph node, blood and spleen. Functional, short‐term in vitro polyclonal stimulation of blood, spleen and lymph node cells from naive mice demonstrated that CCR7^high CD4 T cells produced predominantly interleukin (IL)‐2, whereas CCR7^low CD4 T cells produced both IL‐2 and interferon‐γ (IFN‐γ). However, in contrast to previously published reports, the CCR7^high CD8 T‐cell subpopulation produced both IFN‐γ and IL‐2. Analysis of effector T cells, induced by immunization in vivo, showed that a proportion of activated naïve CD4 T cells down‐regulated CCR7 only after multiple cell divisions, and this coincided with the down‐regulation of CD62L and production of IL‐4 and IFN‐γ. Finally, analysis of effector T cells during the phase of maximal clonal expansion of secondary immune responses in vivo indicated that the vast majority of both IL‐2‐ and IFN‐γ‐producing cells are CCR7^low, while few cytokine‐expressing CCR7^high T cells were detected. Our results support the hypothesis, developed from studies with human cells, that CCR7 may separate functionally different murine memory T‐cell subpopulations, but indicate additional complexity in that CCR7^high CD8 T cells also may produce IFN‐γ.     

Myeloid‐derived suppressor cell activation by combined LPS and IFN‐γ treatment impairs DC development

Myeloid‐derived suppressor cells (MDSC) and DC are major controllers of immune responses against tumors or infections. However, it remains unclear how DC development and MDSC suppressor activity both generated from myeloid precursor cells are regulated. Here, we show that the combined treatment of BM‐derived MDSC with LPS plus IFN‐γ inhibited the DC development but enhanced MDSC functions, such as NO release and T‐cell suppression. This was not observed by the single treatments in vitro. In the spleens of healthy mice, we identified two Gr‐1^lowCD11b^highLy‐6C^highSSC^lowMo‐MDSC and Gr‐1^highCD11b^lowPMN‐MDSC populations with suppressive potential, whereas Gr‐1^highCD11b^high neutrophils and Gr‐1^lowCD11b^highSSC^low eosinophils were not suppressive. Injections of LPS plus IFN‐γ expanded these populations within the spleen but not LN leading to the block of the proliferation of CD8⁺ T cells. At the same time, their capacity to develop into DC was impaired. Together, our data suggest that spleens of healthy mice contain two subsets of MDSC with suppressive potential. A two‐signal‐program through combined LPS and IFN‐γ treatment expands and fully activates MDSC in vitro and in vivo.     

Differential Adhesion Molecule Expression on Porcine Mononuclear Cell Populations

Adhesion molecule expression was analysed on porcine blood and lymphoid organ CD4⁺ CD8^? naïve T helper (Th) lymphocytes, CD4⁺ CD8⁺ memory Th lymphocytes (particular to the pig), CD4^? CD8^high cytotoxic T (Tc) lymphocytes, CD4^? CD8^low NK cells (CD3^? in the pig), CD4^? CD8^? T-cell receptor-γδ-positive (TCRγδ⁺) lymphocytes, B lymphocytes and monocytes. While CD44 expression was relatively homogeneous amongst mononuclear cells, differences were noted for the integrins. Blood naïve Th lymphocytes were CD49d^low CD11a^low, as were splenic naïve Th cells; blood memory Th lymphocytes were CD49d^high CD11a^low, splenic memory Th cells were CD49d^high CD11a^high with a CD49d^high CD11a^low subpopulation; blood Tc lymphocytes were mainly CD49d^low CD11a^low, and splenic cells were CD49d^high CD11a^high. Lymph node lymphocytes were more homogeneous in their integrin expression. These were relatively CD49d^low CD11a^low, except the memory Th lymphocytes which had higher integrin expression. B lymphocytes related to the majority of integrin^low T cells, while monocytes and NK cells were CD49d^high CD11a^high; γδ T lymphocytes showed variable CD49d expression but a CD11a^high phenotype. CD49d^high CD11a^high co-expression was found, and this phenotype was typical of, but not exclusive to, CD25⁺ (activated) lymphocytes. These results demonstrated that porcine memory Th lymphocytes and NK cells, as well as activated cells, would have increased integrin-dependent activities compared with naïve Th lymphocytes, and integrin-dependent reactions would probably vary between blood and lymphoid organ cells.     

Mouse and human CD8+ CD28low regulatory T lymphocytes differentiate in the thymus

Regulatory T (Treg) lymphocytes play a central role in the control of immune responses and so maintain immune tolerance and homeostasis. In mice, expression of the CD8 co‐receptor and low levels of the co‐stimulatory molecule CD28 characterizes a Treg cell population that exerts potent suppressive function in vitro and efficiently controls experimental immunopathology in vivo. It has remained unclear if CD8⁺ CD28^low Treg cells develop in the thymus or represent a population of chronically activated conventional T cells differentiating into Treg cells in the periphery, as suggested by their CD28^low phenotype. We demonstrate that functional CD8⁺ CD28^low Treg cells are present in the thymus and that these cells develop locally and are not recirculating from the periphery. Differentiation of CD8⁺ CD28^low Treg cells requires MHC class I expression on radioresistant but not on haematopoietic thymic stromal cells. In contrast to other Treg cells, CD8⁺ CD28^low Treg cells develop simultaneously with CD8⁺ CD28^high conventional T cells. We also identified a novel homologous naive CD8⁺ CD28^low T‐cell population with immunosuppressive properties in human blood and thymus. Combined, our data demonstrate that CD8⁺ CD28^low cells can develop in the thymus of mice and suggest that the same is true in humans.     

Flt3 ligand‐eGFP‐reporter expression characterizes functionally distinct subpopulations of CD150+ long‐term repopulating murine hematopoietic stem cells

The pool of hematopoietic stem cells (HSCs) in the bone marrow is a mixture of resting, proliferating, and differentiating cells. Long‐term repopulating HSCs (LT‐HSC) are routinely enriched as Lin^?Sca1⁺c‐Kit⁺CD34^?Flt3^?CD150⁺CD48^? cells. The Flt3 ligand (Flt3L) and its receptor Flt3 are important regulators of HSC maintenance, expansion and differentiation. Using Flt3L‐eGFP reporter mice, we show that endogenous Flt3L‐eGFP‐reporter RNA expression correlates with eGFP‐protein expression. This Flt3L‐eGFP‐reporter expression distinguishes two LT‐HSC populations with differences in gene expressions and reconstituting potential. Thus, Flt3L‐eGFP‐reporter^low cells are identified as predominantly resting HSCs with long‐term repopulating capacities. In contrast, Flt3L‐eGFP‐reporter^high cells are in majority proliferating HSCs with only short‐term repopulating capacities. Flt3L‐eGFP‐reporter^low cells express hypoxia, autophagy‐inducing, and the LT‐HSC‐associated genes HoxB5 and Fgd5, while Flt3L‐eGFP‐reporter^high HSCs upregulate genes involved in HSC differentiation. Flt3L‐eGFP‐reporter^low cells develop to Flt3L‐eGFP‐reporter^high cells in vitro, although Flt3L‐eGFP‐reporter^high cells remain Flt3L‐eGFP‐reporter^high. CD150⁺Flt3L‐eGFP‐reporter^low cells express either endothelial protein C receptor (EPCR) or CD41, while Flt3L‐eGFP‐reporter^high cells do express EPCR but not CD41. Thus, FACS‐enrichment of Flt3/ Flt3L‐eGFP‐reporter negative, Lin^?CD150⁺CD48^? EPCR⁺CD41⁺ HSCs allows a further 5‐fold enrichment of functional LT‐HSCs.     

Interleukin‐4‐induced loss of CD8 expression and cytolytic function in effector CD8 T cells persists long term in vivo

Activation of naive CD8⁺ T cells in the presence of interleukin‐4 modulates their CD8 co‐receptor expression and functional differentiation, resulting in the generation of CD8^low cells that produce type 2 cytokines and display poor cytolytic and anti‐tumour activity. Although this CD8^low phenotype becomes stable after about a week and can persist with further stimulation in vitro, it is not known whether it can be maintained long term in vivo. Here we report that CD8^low cells derived from oval‐bumin_257–264‐specific T‐cell receptor‐transgenic CD8⁺ T cells activated in the presence of interleukin‐4 could be detected in the spleen for at least 4 months after adoptive transfer into normal mice. A significant proportion of the long‐term surviving cells retained their CD8^low phenotype in vivo and after clonal re‐activation in vitro. Although long‐term surviving CD8^low cells lacked detectable cytolytic activity or perforin expression, they showed some anti‐tumour function in vivo. The persistence of functional cells with a CD8^low phenotype in vivo raises the possibility that such cells can contribute to effector or regulatory responses to tumours or pathogens.     

AKT蛋白激酶活化对弥漫性大B细胞淋巴瘤细胞生物学行为影响的体外研究

目的观察AKT及磷酸化AKT（pAKT,即AKT呈活化状态））蛋白激酶在三个弥漫性大B细胞淋巴瘤（DLBCL）细胞株中的表达状态,并探讨其与DLBCL细胞生物学行为的关系以及P13K抑制剂LY294002对该瘤细胞生长的影响。方法采用Western blot检测DLBCL细胞株ly1、ly8、ly10在有血清和无血清培养条件下AKT和pAKT的表达状态;用PBK抑制剂LY294002分别作用于三株细胞,观察pAKT水平的改变;用流式细胞术结合碘化丙啶（PI）单染分析细胞周期、Annexin V-异硫氢酸荧光素（FITC）染色检测细胞凋亡、BrdU法观察细胞增殖状态的改变。结果三株DLBCL细胞株在不同的培养条件下均显示有pAKT,在10％FBS培养组ly8和ly10之pAKT水平高于无血清培养组;而ly1相反,在无血清培养组pAKT略高于有血清培养组。在有血清培养组,LY294002可明显降低三细胞株细胞的pAKT水平,并显示S期细胞明显减少（P〈0．05）,G0-G1期细胞增加,增殖细胞比例显著降低（P〈0．05）,以ly8、ly10细胞尤为明显;但凋亡细胞无明显增加（P〉0．05）。结论AKT活化与DLBCL细胞周期和细胞增殖密切相关;与细胞凋亡无显著相关;LY294002可通过降低DLBCL细胞的pAKT水平继而抑制瘤细胞生长。     

Hierarchy of immunosuppressive strength among myeloid‐derived suppressor cell subsets is determined by GM‐CSF

CD11b⁺/Gr‐1⁺ myeloid‐derived suppressor cells (MDSC) contribute to tumor immune evasion by restraining the activity of CD8⁺ T‐cells. Two major MDSC subsets were recently shown to play an equal role in MDSC‐induced immune dysfunctions: monocytic‐ and granulocytic‐like. We isolated three fractions of MDSC, i.e. CD11b⁺/Gr‐1^high, CD11b⁺/Gr‐1^int, and CD11b⁺/Gr‐1^low populations that were characterized morphologically, phenotypically and functionally in different tumor models. In vitro assays showed that CD11b⁺/Gr‐1^int cell subset, mainly comprising monocytes and myeloid precursors, was always capable to suppress CD8⁺ T‐cell activation, while CD11b⁺/Gr‐1^high cells, mostly granulocytes, exerted appreciable suppression only in some tumor models and when present in high numbers. The CD11b⁺/Gr‐1^int but not CD11b⁺/Gr‐1^high cells were also immunosuppressive in vivo following adoptive transfer. CD11b⁺/Gr‐1^low cells retained the immunosuppressive potential in most tumor models. Gene silencing experiments indicated that GM‐CSF was necessary to induce preferential expansion of both CD11b⁺/Gr‐1^int and CD11b⁺/Gr‐1^low subsets in the spleen of tumor‐bearing mice and mediate tumor‐induced tolerance whereas G‐CSF, which preferentially expanded CD11b⁺/Gr‐1^high cells, did not create such immunosuppressive environment. GM‐CSF also acted on granulocyte–macrophage progenitors in the bone marrow inducing local expansion of CD11b⁺/Gr‐1^low cells. These data unveil a hierarchy of immunoregulatory activity among MDSC subsets that is controlled by tumor‐released GM‐CSF.     

CD14lowCD16high: A cytokine-producing monocyte subset which expands during human immunodeficiency virus infection

Infection with the human immunodeficiency virus HIV-1 is associated with the expansion of a CD14^lowCD16^high monocyte subset in peripheral blood. This subset, which represents a minor subpopulation of monocytes in healthy individuals, increases during HIV infection and, in patients with AIDS, may represent up to 40% of the total circulating monocyte cell population. The CD14^lowCD16^high circulating monocytes co-express MAX.1, p150,95 and HLADR which are typical of tissue macrophage markers. These cells also express higher levels of intracellular interleukin (IL)-1α and tumor necrosis factor (TNF)-α than the CD14^highCD16^low monocyte population from the same patients. The CD14^lowCD16^high cells also express low levels of CD35, CD11a and CD4 in common with normal monocytes. When cultured in vitro, monocytes from HIV-seropositive individuals differentiated within a few hours into an elongated fibroblastoid shape characteristic of migratory cells. Our results suggest that the expansion of the CD14^lowCD16^high monocyte subset, which produce high amounts of TNF-α and IL-1α, may participate in the immune dysfunction observed during HIV infection.     

CD14 expression is increased on monocytes in patients with anti‐neutrophil cytoplasm antibody (ANCA)‐associated vasculitis and correlates with the expression of ANCA autoantigens

Monocyte subsets with differing functional properties have been defined by their expression of CD14 and CD16. We investigated these subsets in anti‐neutrophil cytoplasm antibody (ANCA)‐associated vasculitis (AAV) and determined their surface expression of ANCA autoantigens. Flow cytometry was performed on blood from 14 patients with active AAV, 46 patients with AAV in remission and 21 controls. The proportion of classical (CD14^highCD16^neg/low), intermediate (CD14^highCD16^high) and non‐classical (CD14^lowCD16^high) monocytes and surface expression levels of CD14 and CD16 were determined, as well as surface expression of proteinase 3 (PR3) and myeloperoxidase (MPO) on monocyte subsets. There was no change in the proportion of monocytes in each subset in patients with AAV compared with healthy controls. The expression of CD14 on monocytes from patients with active AAV was increased, compared with patients in remission and healthy controls (P < 0·01). Patients with PR3‐ANCA disease in remission also had increased monocyte expression of CD14 compared with controls (P < 0·01); however, levels in patients with MPO‐ANCA disease in remission were lower than active MPO‐ANCA patients, and not significantly different from controls. There was a correlation between CD14 and both PR3 and MPO expression on classical monocytes in AAV patients (r = 0·79, P < 0·0001 and r = 0·42, P < 0·005, respectively). In conclusion, there was an increase in monocyte CD14 expression in active AAV and PR3‐ANCA disease in remission. The correlation of CD14 expression with ANCA autoantigen expression in AAV may reflect cell activation, and warrants further investigation into the potential for increased CD14 expression to trigger disease induction or relapse.     

GRP78 up-regulation is associated with androgen receptor status, Hsp70-Hsp90 client proteins and castrate-resistant prostate cancer

GRP78/BiP is a key member of the molecular chaperone heat shock protein (Hsp) 70 family. It has a critical role in prostate cancer (PC) including Pten loss‐driven carcinogenesis, but the molecular basis of this remains unclear. We investigated the effect of GRP78 and its putative client proteins, including androgen receptor (AR) in clinical PC. Expression of GRP78 and key Hsp70–hsp90 client proteins (HER2, HER3, AR and AKT) were studied in an incidence tissue microarray (TMA) of prostate cancer. The relationship of GRP78 and AR was further tested in in vitro cell models (LNCaP and its derived LNCaP‐CR subclone) and a matched TMA of hormone‐naïve (HNPC) and castrate‐resistant prostate cancer (CRPC). In vitro and in vivo expression of GRP78 and client proteins were assessed by western blotting and immunohistochemistry, respectively, using the weighted histoscore method. Significant co‐expression of GRP78, pAKT, HER2, HER3 and AR was observed in PC. Abnormal AR, GRP78 and pAKT expression have significant impact on patient survival. GRP78 expression in AR⁺ tumours was significantly higher than in AR^? tumours. In keeping with our clinical data, activation of AR by dihydrotestosterone (DHT) potently activated GRP78 expression in both LNCaP and LNCaP‐CR cells. For the first time, using a matched HNPC and CRPC TMA, enhanced cytoplasmic and membranous GRP78 expression was observed in CRPC. Future prospective studies are therefore warranted to validate GRP78 as prognostic marker and therapeutic target, in the context of the AR and pAKT status. In summary, GRP78 is co‐expressed with Hsp70–hsp90 client proteins. Up‐regulated expression of AR and GRP78 expression in untreated prostate cancer predicts a less favourable outcome. This points to the importance of understanding in the molecular interaction among AR, GRP78 and AKT. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Age-related accumulation of LFA-1high cells in a CD8+CD45RAhigh T cell population

The differential expression of CD45 isoforms has been suggested as a marker for stages of post-thymic T cell development, that is, CD45RA+CD45R0^? T cells and CD45RA^?CD45R0⁺ T cells are supposed to be virgin and memory cells respectively. Recently, several adhesion molecules have been shown to be up-regulated on the cell surface of memory T cells, and have been suggested to serve as a memory marker. In this study, we investigated the levels of LFA-1 expression on T cells in various subpopulations defined by CD45 isoform expression in donors of various ages. In CD4⁺ T cells, the proportion of LFA-1^high cells among CD45RA^highCD45R0-T cells remained low in all age groups and did not show significant accumulation with age. CD4⁺CD45RA^?CD45R0^highTcells expressed LFA-1 at a higher level than CD4⁺CD45RA^highCD45R0^? T cells. Thus, the currently prevailing view that CD45RA and CD45R0 can be markers for virgin and primed cells was consistent with LFA-1 expression in CD4⁺ T cell population. In CD8⁺ T cells, however, CD45RA^highCD45R0^? T cells consisted of two distinct subpopulations, LFA-1^low and LFA-1^high cells, whereas CD45RA^?CD45R0^high T cells were almost exclusively LFA-1^high When CD29 expression was examined in place of LFA-1 expression, similar results were obtained; CD45RA^high CD45R0^? T cells consisted of two distinct subpopulations, CD29-^{to low} and CD^29high cells, while CD45RA-CD45R0^high T cells were mostly CD29^high. The proportion of LFA-1^high cells in the CD8+CD45RA^high T cell subpopulation increased significantly as a function of age (r = 0.9, p < 0.001). It ranged from 8% in a 14-year-old donor to 94% in a 79-year-old donor. Furthermore, the proportion of CD8⁺CD45RA^highLFA-1^high cells in the CD8⁺ T cell population increased significantly as a function of age (r = 0.85, p < 0.001). On the other hand, the proportion of LEA-1^high cells in CD8⁺CD45RA^? T cell subpopulation exceeded 90% in most donors irrespective of age. These results indicate that the CD8⁺CD45RA^highCD45R0^? T cell subpopulation contains a considerable number of LFA-1^high cells and CD29^high cells, phenotypically similar to previously activated cells. Thus, in terms of LFA-1 and CD29 expressions, the simple scheme that CD45RA is a marker of virgin cells is not applicable to the CD8⁺ T cell population.     

CD3 expression distinguishes two γδT cell receptor subsets with different phenotype and effector function in tuberculous pleurisy

Tuberculous pleurisy is a naturally occurring site of Mycobacterium tuberculosis (Mtb) infection. Herein, we describe the expression of activation, natural killer (NK) and cell migration markers, as well as effector functions from γδT cells in peripheral blood (PB) and pleural effusion (PE) from tuberculosis patients (TB). We observed a decreased percentage of circulating γδT from TB patients and differential expression of NK as well as of chemokine receptors on PB and PE. Two subsets of γδT cells were differentiated by the CD3/γδT cell receptor (γδTCR) complex. The γδTCR^low subset had a higher CD3 to TCR ratio and was enriched in Vδ2⁺ cells, whereas most Vδ1⁺ cells belonged to the γδTCR^high subset. In PB from TB, most γδTCR^high were CD45RA⁺CCR7^‐ and γδTCR^low were CD45RA^+/?CCR7⁺CXCR3⁺. In the pleural space the proportion of CD45RA^‐CCR7⁺CXCR3⁺ cells was higher. Neither spontaneous nor Mtb‐induced interferon (IFN)‐γ production was observed in PB‐γδT cells from TB; however, PE‐γδT cells showed a strong response. Both PB‐ and PE‐γδ T cells expressed surface CD107a upon stimulation with Mtb. Notably, PE‐γδTCR^low cells were the most potent effector cells. Thus, γδT cells from PB would acquire a further activated phenotype within the site of Mtb infection and exert full effector functions. As γδT cells produce IFN‐γ within the pleural space, they would be expected to play a beneficial role in tuberculous pleurisy by helping to maintain a T helper type 1 profile.     

T Cell Responses and Regulation and the Impact of In Vitro IL‐10 and TGF‐β Modulation During Treatment of Active Tuberculosis

Mycobacterium tuberculosis (Mtb) is particularly challenging for the immune system being an intracellular pathogen, and a variety of T cell subpopulations are activated by the host defence mechanism. In this study, we investigated T cell responses and regulation in active TB patients with drug‐sensitive Mtb (N = 18) during 24 weeks of efficient anti‐TB therapy. T cell activation, differentiation, regulatory T cell (Treg) subsets, Mtb‐induced T cell proliferation and in vitro IL‐10 and TGF‐β modulation were analysed by flow cytometry at baseline and after 8 and 24 weeks of therapy, while soluble cytokines in culture supernatants were analysed by a 9‐plex Luminex assay. Successful treatment resulted in significantly reduced co‐expression of HLA‐DR/CD38 and PD‐1/CD38 on both CD4⁺ and CD8⁺ T cells, while the fraction of CD4⁺CD25^highCD127^low Tregs (P = 0.017) and CD4⁺CD25^highCD127^low CD147⁺ Tregs (P = 0.029) showed significant transient increase at week 8. In vitro blockade of IL‐10/TGF‐β upon Mtb antigen stimulation significantly lowered the fraction of ESAT‐6‐specific CD4⁺CD25^highCD127^low Tregs at baseline (P = 0.047), while T cell proliferation and cytokine production were unaffected. Phenotypical and Mtb‐specific T cell signatures may serve as markers of effective therapy, while the IL‐10/TGF‐β pathway could be a target for early inhibition to facilitate Mtb clearance. However, larger clinical studies are needed for verification before concluding.     

Myeloid cells expressing high level of CD45 are associated with a distinct activated phenotype in glioma

Glioblastoma multiforme is characterized by high accumulation of microglia/macrophages. The function of these tumor-infiltrating myeloid cells is not sufficiently elucidated. Therefore, a better understanding of the precise immune cell composition and function in brain tumors is required. In rodent glioma models, two different myeloid cell populations exist, determined by the expression level of CD45, namely CD11b⁺CD45^low and CD11b⁺CD45^high. Previous analyses of cytokine and marker expression profiles were almost exclusively performed on the entire myeloid cell fraction. Consequently, described pro- and anti-tumoral characteristics were not assigned to the evident subpopulations. In the present study, we used a syngeneic glioblastoma mouse model and subsequent flow cytometric analyses to demonstrate the distinct properties of CD11b⁺CD45^high and the CD11b⁺CD45^low cells. First, the majority of CD11b⁺CD45^high cells expressed high level of GR1 and around 6% of IL10 representing in part features of myeloid-derived suppressor cells, while the CD11b⁺CD45^low fraction displayed no upregulation of these molecules. Second, we detected that specifically the CD11b⁺CD45^high population showed antigen-presenting, co-stimulatory, and inflammatory features. Here, we identified up to 80% of MHCII and approximately 50% of CD86 and TNFα-expressing cells. Investigation of MHCI and CD80 revealed a moderate upregulation. By contrast, in the CD11b⁺CD45^low cell fraction, merely MHCII and TNFα were marginally overexpressed. In summary, these data emphasize the specific phenotype of CD11b⁺CD45^high cells in glioma with suppressive as well as pro-inflammatory characteristics whereas the CD11b⁺CD45^low cells were almost unaffected. Hence, primarily, the subpopulation consisting of CD45^high-expressing cells is activated by the tumor and should be considered as therapeutic target.     

干细胞标志物CD133在神经母细胞瘤中的表达

背景：神经母细胞瘤是婴幼儿和儿童最常见的实体肿瘤之一,靶向肿瘤干细胞的治疗能够有望从根本上治愈肿瘤,但是肿瘤干细胞数量较少,并具有抗凋亡、抗放化疗能力等,导致其对于放疗、化疗并不敏感。 目的：探究干细胞标志物CD133在神经母细胞瘤中的表达及其临床意义。 方法：收集2004年6月至2014年2月新疆医科大学第一附属医院小儿外二科诊治的58例神经母细胞瘤患儿临床资料,其中神经母细胞瘤患儿46例,节细胞母细胞瘤患儿12例,通过免疫组织化学分析法分析所有患儿肿瘤组织中的CD133表达水平,并研究神经母细胞瘤的病理分型、INSS分期、术后存活时长与CD133表达水平之间存在的联系。 结果与结论：共有22例(48%)神经母细胞瘤患儿CD133表达为阳性,有4例(33%)节细胞母细胞瘤患儿CD133表达阳性,肿瘤细胞胞浆为CD133主要表达部位。肿瘤组织各临床分期的CD133阳性率差异明显,其中1,2期为21%,3,4期为64%,4S期为36.4%,差异有显著性意义(P=0.011);组织分型为预后良好型患儿CD133阳性率为29%,明显低于预后不良组织型患儿(57%),差异有显著性意义(P=0.031)。CD133阴性患儿生存周期显著长于CD133阳性患儿(P < 0.05)。结果表明干细胞标志物CD133的表达与神经母细胞瘤发生、发展、转归有密切联系,在评估患儿术后存活时长、改进该病的诊断和治疗等方面具有重要意义。 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程      

Targeting cytokines secreted by CD4+CD25highCD127low regulatory T cells inhibits ovarian cancer progression

Epithelial ovarian cancer (EOC) is one of the major malignant cancers with high rates of early metastasis in which regulatory T cells (Tregs) play an important role. Tregs suppress immune responses and promote the development of tumours in patients with EOC. However, the underlying mechanisms remain unclear. In this study, we found higher levels of CD4⁺CD25^highCD127^low Tregs in patients with EOC than in patients with benign ovarian tumours and healthy donors. The immune inhibitory effect of Tregs functions by maintaining high levels of immunosuppressive cytokines in EOC. The high levels of Tregs and related cytokines (TGF‐β1 or IL‐10) were associated with lymphatic metastasis and FIGO stages of patients with EOC. Expression of matrix metalloproteinase (MMP)‐2 and tissue inhibitors of metalloproteinase (TIMP)‐2 in EOC cell lines were significantly regulated in the coculture experiment with CD4⁺CD25^highCD127^low Tregs sorted from EOC patients. Levels of MMP‐2 and TIMP‐2 conversely changed after blocking IL‐10R and TGF‐β1R in EOC cells. The invasion ability of EOC cells was also significantly downregulated in this process. The metastasis of EOC cells was correlated with the levels of TGF‐β1 or IL‐10. These findings suggested that immunosuppressive cytokines secreted by CD4⁺ Tregs could be a novel target for inhibiting EOC progression.     

SEMA3F prevents metastasis of colorectal cancer by PI3K–AKT‐dependent down‐regulation of the ASCL2–CXCR4 axis

Semaphorin‐3F (SEMA3F), an axonal repulsant in nerve development, has been shown to inhibit the progression of human colorectal cancer (CRC); however, the underlying mechanism remains elusive. In this study we found a negative correlation between the levels of SEMA3F and CXCR4 in CRC specimens from 85 patients, confirmed by bioinformatics analysis of gene expression in 229 CRC samples from the Cancer Genome Atlas. SEMA3F^high/CXCR4^low patients showed the lowest frequency of lymph node and distant metastasis and the longest survival. Mechanistically, SEMA3F inhibited the invasion and metastasis of CRC cells through PI3K–AKT‐dependent down‐regulation of the ASCL2–CXCR4 axis. Specifically, ASCL2 enhanced the invasion and metastasis of CRC cells in vitro and expression of ASCL2 correlated with distant metastasis, tumour size and poor overall survival in CRC patients. Treatment of CRC cells with the CXCR4 antagonist AMD3100 attenuated SEMA3F knockdown‐induced invasion and metastasis of CRC cells in vitro and in vivo. Our study thus demonstrates that SEMA3F functions as a suppressor of CRC metastasis via down‐regulating the ASCL2–CXCR4 axis. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Tumor progression inhibits the induction of multifunctionality in adoptively transferred tumor‐specific CD8+ T cells

It is becoming increasingly evident that the multifunctionality of effector cells at the single‐cell level is an important factor to predict the quality of T‐cell response in immunological protection. The significance of the multifunctionality of T cells in anti‐tumor immunity, however, remains unclear. Here, we assessed the IFN‐γ and TNF‐α production and CD107a mobilization in adoptively transferred tumor‐antigen‐specific CD8⁺ T cells at the single‐cell level. Tumor growth of the murine fibrosarcoma CMS5 was found to limit the induction of multifunctionality in the transferred cells. These cells exhibited insufficient acquisition of the CD25^highGITR^highCD62L^low phenotype and reduced infiltration in tumor. Depletion of Treg facilitated the induction of high multifunctionality of the transferred cells even in the hosts with progressing tumor, leading to enhanced tumor regression. The multifunctionality of the transferred cells correlated with in vivo CTL activity, and T cells with high multifunctionality harvested from hosts with successful therapy induced tumor regression when re‐transferred into the tumor‐bearing hosts. These data suggest that the appearance of multifunctional CD8⁺ effector T cells in vivo is a critical determinant of the success of anti‐tumor immunotherapy and Treg play an important role in the mechanism inhibiting the induction of multifunctionality in effector cells.