Flow cytometry is an information-rich method for cellular analysis. In the age of immunotherapy the importance of flow cytometry in drug development has increased dramatically. When using flow cytometry to generate data for decision making, it is mandatory that the assays and instruments are well characterized. A critical element in assay optimization is the screening of the monoclonal antibodies. In this poster, we will describe the results from PD-1 monoclonal antibody screening where it was observed that the different clones generated different results and consequently different conclusions.
It is generally recognized that the immune checkpoint molecule, programmed cell death protein 1 (PD-1) is expressed at low levels on the surface of resting T and B lymphocytes and is upregulated via signaling through the TCR or B-cell receptor. During the optimization of a PD-1 monitoring flow cytometric method, we observed higher than expected levels of PD-1 expression on resting T cells obtained from healthy donors. The magnitude of this population was both donor and reagent specific.
Five different anti-PD-1 monoclonal antibody clones obtained from different providers were screened (EH12.2H7, EH12.1, eBioJ105, MIH4, D3W4U). They were all titrated to obtain an optimal saturating concentration and then compared. In resting CD4 T cells, the best performance was observed with EH12.2H7 or EH12.1 clones which identified 30-33% of CD4 T cells as PD-1 positive. Clone eBioJ105 staining of the same samples on the same day identified 14 % PD-1 positive CD4 T cells, while clones MIH4 and D3W4U staining indicated no PD-1 expression on CD4 T cells. After ex vivo stimulation with SEB, EH12.2H7, EH12.1 and eBioJ105 clones all yielded similar results (76-80% positive CD4 T cells) but fewer positive cells were identified with MIH4 and none with D3W4U clones.
These results illustrate the importance of thorough reagent evaluation in order to achieve a sensitivity coherent with the information-rich promises of flow cytometry.