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Impact of immunosuppressive and antifungal drugs on PBMC- and whole blood-based flow cytometric CD154+ Aspergillus fumigatus specific T-cell quantification

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Abstract

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.

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Acknowledgements

This work was supported by the Interdisciplinary Centre for Clinical Research (IZKF) Wuerzburg (grant number Z-3/56, SW), by the “Deutsche Forschungsgemeinschaft” (Collaborative Research Center/Transregio 124 “Pathogenic fungi and their human host: Networks of interaction–FungiNet”; project A2 to HE and JL), and by the Bavarian Ministry of Economics, Media, Energy and Technology (grant number BayBIO-1606-003, “T-cell based diagnostic monitoring of invasive aspergillosis in haematological patients” to JL). We would like to thank Dr. Markus Kredel (University Hospital of Wuerzburg) for providing valuable feedback and critical discussion of our data.

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  1. Division of Infectious Diseases, Department of Internal Medicine II, University Hospital of Wuerzburg, Würzburg, Germany

    Lukas Page, Chris D. Lauruschkat, Johanna Helm, Philipp Weis, Maria Lazariotou, Hermann Einsele, Andrew J. Ullmann, Juergen Loeffler & Sebastian Wurster

  2. Infection Control and Employee Health, Department of Infectious Diseases, The University of Texas MD Anderson Cancer Center, 6565 MD Anderson Boulevard, Sheikh Zayed Building, Z8.3002, Houston, TX, 77030, USA

    Sebastian Wurster

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Contributions

AJU, JL, and SW conceived and planned the experiments. LP, CDL, JH, PW, ML, and SW carried out the experiments. LP, CDL, PW, and SW analysed the data. LP and SW wrote the paper. HE contributed to project supervision and manuscript preparation. All authors read and approved the final manuscript.

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Correspondence to Sebastian Wurster.

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Informed consent, approved by the ethics committee of the University of Wuerzburg (105/15 and 42/17), was obtained from all blood donors.

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430_2020_665_MOESM1_ESM.docx

Appendix 1. MIATA reporting modules according to http://miataproject.org. Appendix 2. Gating strategy for Fig. 4 and representative data set. Appendix 3. Approximation of the confidence intervals of antigen-specific T-cell frequencies (DOCX 260 kb)

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Page, L., Lauruschkat, C.D., Helm, J. et al. Impact of immunosuppressive and antifungal drugs on PBMC- and whole blood-based flow cytometric CD154+ Aspergillus fumigatus specific T-cell quantification. Med Microbiol Immunol 209, 579–592 (2020). https://doi.org/10.1007/s00430-020-00665-3

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