Immunotherapies are transforming cancer treatment by harnessing the power of the immune system. Beyond traditional checkpoint inhibitors like CTLA-4, PD-1, and PD-L1, new therapies targeting molecules such as ICOS, IDO-1, LAG-3, OX40, TIM-3, and VISTA are in clinical trials. To enhance clinical care and develop effective combination therapies, it’s crucial to thoroughly characterize the co-expression of immunoregulatory proteins on individual immune cells within the tumor microenvironment.

Development of a 56-Plex Panel

We have designed, developed, optimized, and applied a 56-marker CODEX antibody panel to samples from eight cutaneous T cell lymphoma (CTCL) patients. This panel includes structural, tumor, and immune cell markers, featuring eight immunoregulatory proteins that are either approved or in clinical trials as immunotherapy targets.

This panel allows for extensive immunophenotyping, with 33 of the 56 antibodies recognizing antigens specific to various immune cells, including T cells, B cells, plasma cells, NK cells, macrophages, dendritic cells, Langerhans cells, granulocytes, and mast cells. Our focus was on eight functional immune molecules targeted by approved or trial immunotherapies: ICOS, IDO-1, LAG-3, OX40, PD-1, PD-L1, TIM-3, and VISTA.

Key Findings

Our study revealed significant differences in immunoregulatory protein expression among cell types in CTCL, similar to observations in other tumor types. For instance, PD-1 and ICOS were highly expressed on CD4+ T cells, while TIM-3 and VISTA were predominantly found on macrophages. Additionally, the expression of immunoregulatory proteins varied greatly among CTCL patients; some exhibited high levels of multiple markers, while others showed low levels across the board.

Figure 1. Immune cell phenotyping in CTCL patients using 56-plex spatial proteomics panel. (Fig 3C, Phillips et. al. Front Immunol. 2021)

Figure 2. Functional marker expression on immune cells. (Fig 4C, Phillips et. al. Front Immunol. 2021)