Development of a Flow Cytometry Assay to Predict Immune Checkpoint Blockade-Related Complications

Front Immunol. 2021 Nov 16;12:765644. doi: 10.3389/fimmu.2021.765644. eCollection 2021.


Treatment of advanced melanoma with combined immune checkpoint inhibitor (ICI) therapy is complicated in up to 50% of cases by immune-related adverse events (irAE) that commonly include hepatitis, colitis and skin reactions. We previously reported that pre-therapy expansion of cytomegalovirus (CMV)-reactive CD4+ effector memory T cells (TEM) predicts ICI-related hepatitis in a subset of patients with Stage IV melanoma given αPD-1 and αCTLA-4. Here, we develop and validate a 10-color flow cytometry panel for reliably quantifying CD4+ TEM cells and other biomarkers of irAE risk in peripheral blood samples. Compared to previous methods, our new panel performs equally well in measuring CD4+ TEM cells (agreement = 98%) and is superior in resolving CD4+ CD197+ CD45RA- central memory T cells (TCM) from CD4+ CD197+ CD45RA+ naive T cells (Tnaive). It also enables us to precisely quantify CD14+ monocytes (CV = 6.6%). Our new "monocyte and T cell" (MoT) assay predicts immune-related hepatitis with a positive predictive value (PPV) of 83% and negative predictive value (NPV) of 80%. Our essential improvements open the possibility of sharing our predictive methods with other clinical centers. Furthermore, condensing measurements of monocyte and memory T cell subsets into a single assay simplifies our workflows and facilitates computational analyses.

Keywords: assay validation; biomarker; effector memory T cells; flow cytometry; immune checkpoint inhibition; immune-related adverse events; monocytes; prediction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aged
  • Aged, 80 and over
  • CD4-Positive T-Lymphocytes / immunology
  • Female
  • Flow Cytometry / methods*
  • Humans
  • Immune Checkpoint Inhibitors / adverse effects*
  • Male
  • Melanoma / drug therapy*
  • Memory T Cells / immunology
  • Middle Aged
  • Seasons


  • Immune Checkpoint Inhibitors