immune-modulating抗癌的功效apeutic antibodies that have been FDA-approved in recent years, such as anti-CTLA-4 and anti-PD-1, has driven growing interest in methods that provide a mechanistic understanding of drug function. Development of new mono and combination therapies with immune-modulatory effects requires more powerful immunophenotyping techniques capable of in depth cell characterization. Flow-cytometry-based functional assays can be essential for elucidating the mechanism of a drug. While standard flow cytometry can identify immune subsets and their relative abundance within a sample, functional assays allow for the characterization of the biological significance of these populations including suppressive capability or activation status of a target population.
Functional assays usually involve ex vivo stimulation of cells with drugs, antibodies, and/or cytokines, followed by staining for markers in order to quantify a biological response. Covance has validated functional assays that allow effective characterization of immune subsets, including:
- 混合淋巴细胞反应（MLRS）：来自两只小鼠的两个同种异体菌株的脾细胞，刺激群和响应者群体是共同培养的。用CFSE染色测量响应者群的增殖，这是一种胺反应性染料，允许跟踪多达七个循环的细胞分裂。来自肿瘤 - 幼稚刺激动物的脾细胞引发增殖，而轴承某种类型肿瘤的动物的脾细胞将抑制增殖。该测定可用于测量药物治疗引起的免疫抑制的改变。下面的数据代表来自A20-肿瘤小鼠的穗细胞作为刺激细胞时，MLR中增殖的抑制。
- NK细胞刺激：脾细胞或解离肿瘤用IL-12和IL-18培养，以防止产生的细胞因子排泄。量化包括IFN-γ的标记的表达以在总NK细胞群内鉴定活化的NK细胞。
- B-cell Stimulation: Splenocytes or dissociated tumors are cultured with IgM and expression of markers including CD69 are quantified to identify activated B cells within the total B cell population.
- T-cell Stimulation: Splenocytes can be cultured with IL-2, anti-CD28, and anti-CD3e or a drug of interest after CFSE staining. After stimulation, differential rates of proliferation and survival can be measured. The below data represent proliferation in culture with (red) and without (blue) antibody stimulation.
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