CTLA-4 Immunohistochemistry and Quantitative Image Analysis for Profiling of Human Cancers

There is an important need in immuno-oncology to develop reliable immunohistochemistry (IHC) to assess the expression of CTLA-4⁺ tumor-infiltrating lymphocytes in human cancers and quantify them with image analysis (IA). We used commercial polyclonal and monoclonal antibodies and characterized three chromogenic cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) assays with suitable specificity and sensitivity for use in formalin-fixed, paraffin-embedded (FFPE) tissues. We found variable numbers of CTLA-4⁺ lymphocytes in multiple types of cancer and secondary lymphoid organs (SLOs) and other normal human tissues. Combining CTLA-4 with CD3, CD4, or CD8 by immunofluorescence showed that CTLA-4⁺ lymphocytes in SLOs and tumors were typically CD3⁺ and CD4⁺, but not CD8⁺. Individual lymphocytes expressed CTLA-4 either as primarily granular cytoplasmic staining or as excentric globular deposits. The CTLA-4/FoxP3 (forkhead box P3 protein) duplex IHC demonstrated that CTLA-4⁺/FoxP3^- lymphocytes predominated in the germinal centers of SLOs and tumor tertiary lymphoid structures (TLSs), whereas CTLA-4⁺/FoxP3⁺ lymphocytes populated the T-cell zone of SLOs and TLSs, plus tumor stroma. IA scoring was highly comparable with pathologist scoring for CTLA-4 and CTLA-4/FoxP3 assays and a FoxP3 single IHC. Our findings show that CTLA-4 IHC can be used to reliably label lymphocytes in FFPE human tissues, making it possible to investigate the role of CTLA-4 in the tumor microenvironment.