Impact of immunosuppressive and antifungal drugs on PBMC- and whole blood-based flow cytometric CD154 + Aspergillus fumi
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ORIGINAL INVESTIGATION
Impact of immunosuppressive and antifungal drugs on PBMC‑ and whole blood‑based flow cytometric CD154+ Aspergillus fumigatus specific T‑cell quantification Lukas Page1 · Chris D. Lauruschkat1 · Johanna Helm1 · Philipp Weis1 · Maria Lazariotou1 · Hermann Einsele1 · Andrew J. Ullmann1 · Juergen Loeffler1 · Sebastian Wurster1,2 Received: 25 September 2019 / Accepted: 14 March 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Flow cytometric quantification of C D154+ 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 C D154+ 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 costimulation 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. Keywords Antifungals · GvHD prophylaxis · Aspergillus · Biomarker · Flow cytometry · CD49d
Edited by Volkhard A. J. Kempf. Parts of this study have been presented at the European Congress of Clinical Microbiology and Infectious Diseases 2016, Amsterdam, the Netherlands. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00430-020-00665-3) contains supplementary material, which is available to authorized users. * Sebastian Wurster [email protected] 1
Division of Infectious Diseases, Department of Internal Medicine II, University Hospital of Wuerzburg, Würzburg, Germany
Infection Control and Employee Health, Department of Infectious Diseases, The University of Texas MD Anderson Cancer Center,
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