Drug Safety Research and Development, Pfizer Global Research and Development, Groton, CT 06340, USA.
J Immunotoxicol. 2010 Oct-Dec;7(4):357-66. doi: 10.3109/1547691X.2010.523881. Epub 2010 Nov 11.
Preclinical immunotoxicity assessments may be performed during pharmaceutical drug development in order to identify potential cause for concern prior to use in the clinic. The in vivo T-dependent antibody response (TDAR) is widely used in this regard, given its sensitivity to known immunosuppressive compounds, but may be impractical early in drug development where quantities of test article are limited. The goal of the current work is to develop an in vitro human cell-based assay that is sensitive to immunosuppression, uses relatively small quantities of test article, and is simple to perform with moderate to high throughput. Ideally, this assay would require the cooperation of multiple cellular compartments to produce a response, similar to the TDAR. Although the Mishell-Dutton assay (in vitro mouse splenic sheep red blood cell response) has been used for this purpose, it shows considerable inter-laboratory variability, and rodent cells are used which leads to potential difficulty in translation of findings to humans. We have developed an assay that measures an influenza antigen-specific response using frozen-stored human peripheral blood mononuclear cells, which we have termed the human lymphocyte activation (HuLA) assay. The HuLA assay is sensitive to cyclosporine, dexamethasone, rapamycin, mycophenolic acid, and methotrexate at concentrations within their respective therapeutic ranges. Although proliferation is the primary endpoint, we demonstrate that flow cytometry approaches may be used to characterize the proliferating lymphocyte subsets. Flu antigen-specific proliferation in the HuLA assay primarily involves both CD4+ and CD8+ T-lymphocytes and B-lymphocytes, although other lymphocyte subsets also proliferate. In addition, flu-specific antibody-secreting cells can be measured in this assay by ELISPOT, a response that is also sensitive to known immunosuppressive compounds. The HuLA assay represents a relatively straightforward assay with the capability of detecting immune suppression in human cells and can be applied to compound ranking and immunotoxicity assessment.
临床前免疫毒性评估可在药物开发过程中进行，以便在临床使用前发现潜在的关注原因。体内 T 依赖性抗体反应 (TDAR) 在这方面得到了广泛应用，因为它对已知的免疫抑制化合物敏感，但在药物开发的早期阶段，由于测试物质的数量有限，可能不太实际。目前的工作目标是开发一种对免疫抑制敏感的基于人类细胞的体外测定法，该方法使用相对较少的测试物质，并且操作简单，具有中等至高通量。理想情况下，该测定法需要多个细胞区室的合作才能产生反应，类似于 TDAR。尽管 Mishell-Dutton 测定法（体外鼠脾绵羊红细胞反应）已用于此目的，但它显示出相当大的实验室间变异性，并且使用的是啮齿动物细胞，这导致发现难以转化为人类。我们已经开发了一种使用冷冻储存的人外周血单核细胞测量流感抗原特异性反应的测定法，我们将其称为人类淋巴细胞激活（HuLA）测定法。HuLA 测定法对环孢素、地塞米松、雷帕霉素、霉酚酸和甲氨蝶呤的浓度在其各自的治疗范围内敏感。尽管增殖是主要终点，但我们证明可以使用流式细胞术方法来表征增殖的淋巴细胞亚群。HuLA 测定法中的流感抗原特异性增殖主要涉及 CD4+和 CD8+T 淋巴细胞和 B 淋巴细胞，尽管其他淋巴细胞亚群也增殖。此外，通过 ELISPOT 可以测量该测定法中的流感特异性抗体分泌细胞，该反应也对已知的免疫抑制化合物敏感。HuLA 测定法是一种相对简单的测定法，具有检测人细胞中免疫抑制的能力，并可用于化合物分级和免疫毒性评估。
