Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1⁺ Tim-3⁺ CD8⁺ T Cells

Abstract

Immune cells are important components of the tumor microenvironment and influence tumor growth and evolution at all stages of carcinogenesis. Notably, it is now well established that the immune infiltrate in human tumors can correlate with prognosis and response to therapy. The analysis of the immune infiltrate in the tumor microenvironment has become a major challenge for the classification of patients and the response to treatment. The co-expression of inhibitory receptors such as Program Cell Death Protein 1 (PD1; also known as CD279), Cytotoxic T Lymphocyte Associated Protein 4 (CTLA-4), T-Cell Immunoglobulin and Mucin Containing Protein-3 (Tim-3; also known as CD366), and Lymphocyte Activation Gene 3 (Lag-3; also known as CD223), is a hallmark of T cell exhaustion. We developed a multiparametric in situ immunofluorescence staining to identify and quantify at the cellular level the co-expression of these inhibitory receptors. On a retrospective series of frozen tissue of renal cell carcinomas (RCC), using a fluorescence multispectral imaging technology coupled with an image analysis software, it was found that co-expression of PD-1 and Tim-3 on tumor infiltrating CD8 T cells is correlated with a poor prognosis in RCC. To our knowledge, this represents the first study demonstrating that this automated multiplex in situ technology may have some clinical relevance.

In situ multiplex immunofluorescence analysis of the inflammatory burden in kidney allograft rejection: A new tool to characterize the alloimmune response

“…The 45 renal biopsies were analyzed using the software inForm Tissue Finder 2.3.0 (PerkinElmer). Only scanned images at ×20 (ROI fields) were analyzed by inForm . This software is based on a supervised machine‐learning algorithm.…”

The prospect of targeting T cell immunoglobulin and mucin‐domain containing‐3 in renal cell carcinoma immunotherapy

“…These findings indicate that infiltrated Tim‐3 + PD1 + CD8 + T cells are potential mediators to create an aggressive phenotype in patients with RCC 110 . It was also reported that co‐expression of Tim‐3 and PD‐1 on tumour‐infiltrating CD8 + T cells was associated with a poor prognosis in RCC patients 111 . CD4 + and CD8 + T cells of TIL in renal cell carcinoma patients have higher frequencies of Tim‐3 + , PD‐1 + and LAG‐3 + antigens 112 .…”

Presence of Tim3+ and PD‐1+CD8+T cells identifies microsatellite stable colorectal carcinomas with immune exhaustion and distinct clinicopathological features

“…Our data point to a favorable outcome for MSS‐ImmEx hi tumors that have high proportions of Tim‐3 + PD‐1 + CD8 + TILs. This is in contrast to renal cell carcinoma, where high proportions of such cells are associated with poor prognosis [41,42]. Furthermore, CD8 + T cells expressing PD‐1 but not Tim‐3 correlated with higher progression‐free survival and objective response rates to anti‐PD‐1 therapy in renal cell cancer [43].…”