| Adoptive Transfer of Tumor Expanded Regulatory T Cells (Tregs)
|
|
Author:
Date:
2016-08-20
[Abstract] Regulatory T cells (Tregs), a subset of CD4+CD25+ T cells, infiltrate tumors and suppress antitumor activity of effector T and NK cells. Depletion of Tregs by anti CD25+ antibodies has been shown to reduce tumor growth and metastasis (Olkhanud et al., 2009). Conversely, adoptive transfer of Tregs induced immune suppression and promoted tumor growth (Smyth et al., 2006; Janakiram et al., 2015). We have adoptively transferred Tregs to evaluate their immunosuppressive function in vivo. Our study (Vences-Catalan et al., 2015) compared the immunosuppressive efficacy of Tregs derived from tumor-bearing wild type to those of CD81KO mice. The following protocol could be adapted to any other source of Tregs.
Lymph ...
[摘要] 调节性T细胞（Treg）（CD4 + CD25 + T细胞的子集）浸润肿瘤并抑制效应T和NK细胞的抗肿瘤活性。已显示抗CD25 sup/+抗体减少Treg减少肿瘤生长和转移（Olkhanud等人，2009）。相反，Treg的过继转移诱导免疫抑制并促进肿瘤生长（Smyth等人，2006; Janakiram等人，2015）。我们已经过继转移Treg以评价它们在体内的免疫抑制功能。我们的研究（Vences-Catalan等人，2015）比较了源自荷瘤野生型的Treg与CD81KO小鼠的免疫抑制功效。以下协议可以适用于任何其他来源的Tregs。
通过使用来自MACS Miltenyi Biotec的CD4 + CD25 +调节性T细胞分离试剂盒的两步程序分离和纯化淋巴结或脾肿瘤诱导的Treg。首先，通过阴性选择，然后CD25阳性选择+ T细胞富集CD4 + T细胞。然后将肿瘤诱导的纯化的Treg（CD3 + CD4 CD25 + FoxP3 + 与肿瘤细胞转化为幼稚小鼠（Winn测定）（Winn，1960）。根据研究需要，Treg也可以静脉内注射一次或几次。然后通过测径器或通过体内成像技术测量过继转移的Treg对肿瘤生长的影响。
|
|
|
| Macrophage Polarization by Tumor-induced MDSCs Assay
|
|
Author:
Date:
2016-08-20
[Abstract] Myeloid derived suppressor cells (MDSCs) are a subset of granulocytes (immature myeloid cells) that exploit a variety of mechanism to modulate the innate and adaptive immune system. MDSCs are present normally in the body, but their numbers increase during inflammation and in cancer, promoting an immunosuppressive microenvironment. In addition to MDSCs, macrophages also play an important role during cancer development. There are two subsets of tumor associated macrophages (TAMs): M1 and M2. M1 are “anti-tumor” macrophages that are activated by interferon gamma (IFN-γ) and/or Lipopolysaccharide (LPS) and secrete high amount of interleukin 12 (IL-12) thereby inducing a Th1 anti-tumor immune response. M2 or “pro-tumorigenic” macrophages are activated by interleukin 4 (IL-4) and interleukin 10 ...
[摘要] 骨髓衍生的抑制细胞（MDSC）是粒细胞（未成熟骨髓细胞）的子集，其利用多种机制调节先天和适应性免疫系统。 MDSC通常存在于体内，但它们的数量在炎症和癌症期间增加，从而促进免疫抑制微环境。除了MDSC，巨噬细胞也在癌症发展中发挥重要作用。肿瘤相关巨噬细胞（TAM）有两个亚类：M1和M2。 M1是由干扰素γ（IFN-γ）和/或脂多糖（LPS）活化并分泌大量白细胞介素12（IL-12）从而诱导Th1抗肿瘤免疫应答的"抗肿瘤"巨噬细胞。 M2或"致肿瘤发生"巨噬细胞被白介素4（IL-4）和白细胞介素10（IL-10）激活并分泌大量的IL-10，这促进肿瘤进展（Gabrilovich等人， 。，2012）。在肿瘤微环境中MDSC和巨噬细胞之间的相互作用显示增强这些亚群介导的免疫抑制。 MDSC通过产生IL-10而影响TAM，其继而诱导IL-12的下调并将M1极化为M2巨噬细胞。在我们的研究中，我们使用以下方案来评价肿瘤诱导的MDSC将LPS活化的M1极化为M2巨噬细胞的能力（Vences-Catalan等人，2015）。该方案改编自先前的研究（Sinha等人，2007）。
|
|
|
| HIV-1 Fusion Assay
|
|
Author:
Date:
2014-08-20
[Abstract] The HIV-1 fusion assay measures all steps in the HIV-1 life cycle up to and including viral fusion. It relies on the incorporation of a β-lactamase–Vpr (BlaM-Vpr) protein chimera into the virion and the subsequent transfer of this chimera into the target cell by fusion (Figure 1). The transfer is monitored by the enzymatic cleavage of CCF2, a fluorescent dye substrate of β-lactamase, loaded into the target cells. Cleavage of the β-lactam ring in CCF2 by β-lactamase changes the fluorescence emission spectrum of the dye from green (520 nm) to blue (447 nm). This change reflects virion fusion and can be detected by flow cytometry (Figure 2).
[摘要] HIV-1融合测定测量HIV-1生命周期中直至并包括病毒融合的所有步骤。 它依赖于将β-内酰胺酶-Vpr（BlaM-Vpr）蛋白嵌合体并入病毒体中，并且随后通过融合将该嵌合体转移到靶细胞中（图1）。 通过CCF2（一种β-内酰胺酶的荧光染料底物）的酶裂解来监测转移，将其装载到靶细胞中。 β-内酰胺酶在CCF2中的β-内酰胺环的切割将染料的荧光发射光谱从绿色（520nm）改变为蓝色（447nm）。 这种变化反映了病毒体融合，并且可以通过流式细胞术检测（图2）。
|
|
|