PD-1(+)CD8(+) T Cells Proximal to PD-L1(+)CD68(+) Macrophages Are Associated with Poor Prognosis in Pancreatic Ductal Adenocarcinoma Patients

SIMPLE SUMMARY: The current methods for assessing tumor microenvironments (TMEs) using cellular markers and cell density-based assays do not identify the primitive phenotypes of single cells with multilineage selectivity or functional or cellular spatial information in tissues. Here, we combined the strategy of multiplex IHC imaging and cytometry-based cell quantification to assess multiple lineage-selective and functional phenotypic biomarkers and deeply dissect the state of immune complexity in the TME in association with clinical outcomes. Our findings showed that the percentage of CD8(+) T cells expressing the T cell exhaustion marker PD-1 and/or the high expression of the checkpoint PD-L1 among CD68(+) cells was associated with poor prognosis. The prognostic value of this combined approach is greater than that of lymphocyte and myeloid cell density analyses. Furthermore, a spatial analysis revealed that the abundance of PD-L1(+)CD68(+) tumor-associated macrophages correlated with PD-1(+)CD8(+) T cell infiltration levels. Our findings further strengthen the evidence for applying IHC imaging and cytometry-based cell analyses in precision medicine. ABSTRACT: Immune complexity status in the TME has been linked to clinical outcomes in pancreatic ductal adenocarcinoma (PDAC) patients. TME assessments with current cell marker and cell density-based analyses do not identify the original phenotypes of single cells with multilineage selectivity, the functional status of the cells, or cellular spatial information in the tissues. Here, we describe a method that circumvents these problems. The combined strategy of multiplexed IHC with computational image cytometry and multiparameter cytometric quantification allows us to assess multiple lineage-selective and functional phenotypic biomarkers in the TME. Our study revealed that the percentage of CD8(+) T lymphoid cells expressing the T cell exhaustion marker PD-1 and the high expression of the checkpoint PD-L1 in CD68(+) cells are associated with a poor prognosis. The prognostic value of this combined approach is more significant than that of lymphoid and myeloid cell density analyses. In addition, a spatial analysis revealed a correlation between the abundance of PD-L1(+)CD68(+) tumor-associated macrophages and PD-1(+)CD8(+)T cell infiltration, indicating pro-tumor immunity associated with a poor prognosis. These data highlight the implications of practical monitoring for understanding the complexity of immune cells in situ. Digital imaging and multiparameter cytometric processing of cell phenotypes in the TME and tissue architecture can reveal biomarkers and assessment parameters for patient stratification.