Flow cytometric quantification of CD154⁺ mould specific T-cells in antigen-stimulated peripheral blood mononuclear cells (PBMCs) or whole blood has been described as a supportive biomarker to diagnose invasive mould infections and to monitor therapeutic outcomes. As patients at risk frequently receive immunosuppressive and antifungal medication, this study compared the matrix-dependent impact of representative drugs on CD154⁺ T-cell detection rates. PBMCs and whole blood samples from healthy adults were pre-treated with therapeutic concentrations of liposomal amphotericin B, voriconazole, posaconazole, cyclosporine A (CsA) or prednisolone. Samples were then stimulated with an Aspergillus fumigatus lysate or a viral antigen cocktail (CPI) and assessed for CD154⁺ T-helper cell frequencies. Specific T-cell detection rates and technical assay properties remained largely unaffected by exposure of both matrices to the studied antifungals. By contrast, CsA and prednisolone pre-treatment of isolated PBMCs and whole blood adversely impacted specific T-cell detection rates and caused elevated inter-replicate variation. Unexpectedly, the whole blood-based protocol that uses additional α-CD49d co-stimulation was less susceptible to CsA and prednisolone despite prolonged drug exposure in the test tube. Accordingly, addition of α-CD49d during PBMC stimulation partially attenuated the impact of immunosuppressive drugs on test performance. Translating these results into the clinical setting, false-negative results of CD154⁺ antigen-specific T-cell quantification need to be considered in patients receiving T-cell-active immunosuppressive medication. Optimized co-stimulation regimes with α-CD49d could contribute to an improved feasibility of functional T-cell assays in immunocompromised patient populations.

中文翻译：

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免疫抑制和抗真菌药物对PBMC和全血流式细胞仪CD154 +烟曲霉特异性T细胞定量的影响。

抗原刺激的外周血单核细胞（PBMC）或全血中CD154 ⁺霉菌特异性T细胞的流式细胞术定量已被描述为诊断侵入性霉菌感染和监测治疗结果的支持性生物标志物。由于高危患者经常接受免疫抑制和抗真菌药物，本研究比较了代表性药物对CD154 ⁺ T细胞检出率的基质依赖性影响。用治疗浓度的脂质体两性霉素B，伏立康唑，泊沙康唑，环孢素A（CsA）或泼尼松龙对健康成人的PBMC和全血样品进行预处理。然后用烟曲霉刺激样品裂解物或病毒抗原混合物（CPI）并评估CD154 ⁺T辅助细胞频率。两种基质都暴露于研究的抗真菌剂中，特定的T细胞检测率和技术分析特性仍未受到很大影响。相比之下，CsA和泼尼松龙对分离的PBMC和全血的预处理会对特异性T细胞检测率产生不利影响，并导致复制间变异增加。出乎意料的是，尽管在试管中的药物暴露时间延长，但使用额外的α-CD49d共刺激的全血方案对CsA和泼尼松龙的敏感性较低。因此，在PBMC刺激过程中添加α-CD49d会部分减弱免疫抑制药物对测试性能的影响。将这些结果转化为CD154 ⁺的临床阴性假结果接受T细胞活性免疫抑制药物的患者需要考虑抗原特异性T细胞定量。与α-CD49d共同优化的治疗方案可以改善免疫功能低下患者群体中功能​​性T细胞测定的可行性。