Cell Processing and Immune Assessment

该计划研究项目的中心主题是骨髓瘤肿瘤细胞的生长通过与肿瘤细胞微环境的多种功能多样的相互作用来调节。许多这些相互作用涉及先天性和适应性免疫的元素，可以促进或抑制骨髓瘤细胞在体内的生长。更好地了解免疫系统的负性和正性相互作用可以为骨髓瘤患者开发创新疗法。这是3个研究项目中每个项目的目标，该核心将通过提供用于免疫监测和制造用于过继细胞治疗的免疫细胞的集中实验室资源来支持每个项目。 对于接受常规治疗的骨髓瘤患者以及参加本项目支持的特定临床试验的患者，我们已经建立了获得骨髓瘤肿瘤细胞和正常免疫细胞样本用于研究的程序（见核心A）。在知情同意后获得骨髓抽吸物和活组织检查以及外周血细胞，并定期冷冻保存活细胞、血浆和血清。使用这些样本，核心C将使用各种方法提供免疫功能的详细分析，这些方法提供特定人群的定量评估， 以及它们的成熟和功能水平。循环免疫细胞以及骨髓细胞和骨髓瘤细胞的定量分析通过多参数流式细胞术用一组荧光染料缀合的单克隆抗体测定。免疫细胞的不同亚群的细胞因子产生也通过流式细胞术测定。通过基于珠的多重测定或通过ELISA测量单个细胞因子的水平。T细胞受体库的重建通过TCR VB光谱分析来检查。通过T细胞受体切除环（TREC）的定量PCR评价胸腺功能。通过ELISPOT或用荧光HLA肽缀合物测定对特异性靶抗原如CMV和EBV或骨髓瘤相关肿瘤抗原如MUC-1的T细胞免疫。这些测定的结果与免疫功能的其他参数以及临床结果相关。对于入组项目3中描述的骨髓瘤/DC融合疫苗和T细胞输注临床试验的患者，该核心将额外负责临床规模生产用于过继性T细胞输注的大量自体T细胞。该核心的具体目标如下： 1.提供免疫功能和对免疫治疗反应的表型和功能测量。 2.生产用于过继免疫治疗的患者T细胞

A central theme of the research projects in this program is that the growth of myeloma tumor cells is modulated through multiple and functionally diverse interactions with the tumor cell microenvironment. Many of these interactions involve elements of innate and adaptive immunity that can either promote or inhibit the growth of myeloma cells in vivo. A better understanding of both negative and positive immune system interactions can lead to the development of innovative therapies for patients with myeloma. This is a goal of each of the 3 research projects and this core will support each project by providing a centralized laboratory resource for immune monitoring and manufacturing immune cells for adoptive cellular therapy. For patients with myeloma who receive conventional therapies as well as those enrolled on specific clinical trials supported by this program, we have established procedures for obtaining samples of myeloma tumor cells and normal immune cells for research studies (described in Core A). Bone marrow aspirates and biopsies and well as peripheral blood cells are obtained after informed consent and viable cells, plasma and serum are cryopreserved at regular intervals. Using these samples Core C will provide a detailed analysis of immune function using a variety of methods that provide a quantitative assessment of specific populations as well as their level of maturation and function. Quantitative analysis of circulating immune cells as well as bone marrow cells and myeloma cells is determined by multi-parameter flow cytometry with a panel of fluorochrome-conjugated monoclonal antibodies. Cytokine production by distinct subsets of immune cells is also determined by flow cytometry. Levels of individual cytokines are measured by bead-based multiplex assays or by ELISA. Reconstitution of T cell receptor repertoire is examined by TCR VB spectratyping. Thymic function is evaluated by quantitative PCR for T-cell receptor excision circles (TREC). T cell immunity to specific target antigens such as CMV and EBV or to myeloma-associated tumor antigens such as MUC-1 is determined by ELISPOT or with fluorescent-HLA-peptide-conjugates. Results of these assays are correlated with other parameters of immune function as well as with clinical outcomes. For patients enrolled on the clinical trials of myeloma/DC fusion vaccine and T cell infusions described in Project 3, this core will have the additional responsibility for clinical scale manufacturing of large numbers of autologous T cells for adoptive T cell infusions. The specific aims of this core are listed below: 1. To provide phenotypic and functional measurements of immune function and response to immune therapies. 2. Manufacture patient T cells for adoptive immune therapy