Tumor-associated immune suppression can lead to defective T-cell mediated antitumor immunity. CD8 T-cells play a critical role in the host defense against cancers and infectious diseases. However, the presence of antigen-specific CD8 T-cells does not always imply that cancers and/or pathogens are efficiently eliminated in the body. In tumor infiltrating lymphocytes (TILS), other markers including CD103, PD-1 and FOXP3 are broadly expressed and have shown a wider range of immunoregulatory and important roles in T-cell activation, T- cell regulatory and programmed cell-death checkpoints. Existing methods can either deliver phenotypic information on homogenous samples such as flow cell cytometry or give immunohistochemical information on single biomarker. However, it would be of advantage to be able to visualize the distributions of multiple biomarkers in TILs that would discriminate stromal cells versus solid tumor. Therefore, a multiplex IHC stain utilizing a melanoma marker as a staining mask that separates the malignant tumor cells from stromal cells maybe a good strategy to facilitate better interpretation.

Materials and Methods

Monoclonal mouse antibodies to CD8, FOXP3 and PD-1 and rabbit monoclonal antibodies CD8, CD103 and PD-1 were optimally tittered for single, double and triple stains and visualized with HRP DAB/Black chromogens and/or alkaline phosphatase fast red/blue chromogens. A mouse monoclonal pan melanoma cocktail was visualized with a fast red chromogen and was used as a staining mask for melanoma tumors cells, but not stromal cells.


Single, double and triple stains that included pan melanoma cocktail (mask) and CD8, CD103 FOXP3 and PD-1 were successfully established. All double and triple stains gave comparable results to single stains. The co-expression of TILs including CD8 and CD103 or CD8 or FOXP3 and PD-1 was easily identified with contrasting chromogens. TILs in stroma tissues and in malignant tumors cells were easily discriminated with the pan melanoma (mask) that stained only tumor cells with fast red chromogen, but did not stain TILS or stromal tissues.


The evaluation of TILS in malignant melanoma was enhanced with multiplex stains. TILs in stromal tissues and melanoma tumors cells were easily distinguished with the Pan Melanoma staining mask. These finding may help facilitate important prognostic information and thus may support the rationale for using immunotherapy strategies.

Citation Format: David E. Tacha, Wei Yuan, Jillian Terrell. Immunohistochemical multiplex staining strategies with CD8, CD103, PD-1, FOXP3 and pan melanoma cocktail in tumor infiltrating lymphocytes in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 467.