Case Study

Bystander CD8+ T cells are abundant and phenotypically distinct in human tumor infiltrates

Yannick Simoni, Etienne Becht, Michael Fehlings, Chiew Yee Loh, Si-Lin Koo, Karen Wei Weng Teng, Joe Poh Sheng Yeong, Rahul Nahar, Tong Zhang, Hassen Kared, Kaibo Duan, Nicholas Ang, Michael Poidinger, Yin Yeng Lee, Anis Larbi, Alexis J. Khng, Emile Tan, Cherylin Fu, Ronnie Mathew, Melissa Teo, Wan Teck Lim, Chee Keong Toh, Boon-Hean Ong, Tina Koh, Axel M. Hillmer, Angela Takano, Tony Kiat Hon Lim, Eng Huat Tan, Weiwei Zhai, Daniel S. W. Tan, Iain Beehuat Tan & Evan W. Newell

CD8+ T cells harbour specific T cell receptors (TCRs) that recognize cognate antigens as small peptides bound to major histocompatibility complex (MHC) class I molecules. Tumor-specific antigens are expressed exclusively by cancer cells and neoantigens are a subset that arise from somatic mutations. There is strong evidence that tumor regression may occur in some melanoma or non-small lung cell carcinoma patients during checkpoint blockade therapy through the  elicitation of tumor-specific T cell responses (1-6), which are directed toward unique, patient-specific neoantigens (7-8). However, there are great challenges associated with accurately predicting and identifying neoantigens for therapeutic purposes. In this study, we used mass cytometry in combination with combinatorial MHC-tetramer multiplexing to screen and identify the antigen-specificity of tumor-reactive and cancer-unrelated (“bystander”) tumor infiltrating lymphocytes (TILs) and their phenotypes, in order to gain insight into the tumor immune composition, which might inform on future therapeutic intervention strategies.

This study demonstrates that CD8+ TILs from colon and lung tumors are not all specific for tumor antigens, but may include “bystander” CD8+ TILs specific for cancer-unrelated epitopes possessing a wide range of frequencies and phenotypes. High frequencies of CD39+ neoantigen-specific CD8+ T cells were found in the tumor compared with tumor-unrelated CD8+ T cells, which were characterized by an absence of CD39 expression. (fig. 1). Interestingly, CD39+ CD8+ TILs displayed reduced diversity of TCR sequences, suggesting that these cells had undergone tumor-antigen-driven clonal expansion and displayed characteristics of exhausted cells, while conversely, an absence of CD39 in CD8+ TILs defined populations that lack hallmarks of chronic antigen stimulation at the tumour site, supporting their classification as bystanders. Together, these results indicate that CD39 could be useful as a marker of tumor-specific CD8+ T cells, which could be exploited for the development of novel biomarkers or therapeutics.

Figure 1. Summary figure for the study. CD8+ TILs are not only specific for tumor-specific antigens but may also contain populations that recognize cancer-unrelated epitopes such as viral epitopes. These cancer-unrelated TILs were shown to lack the expression of CD39.

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Peripheral blood mononuclear cells (PBMC) and tumor samples were obtained from patients with colorectal or lung cancer. Using whole exome and RNA sequencing we identified tumor neoantigens that are predicted to bind to MHC-I. We utilized mass cytometry, together with specific antibody staining panel consisting of lineage, descriptive and specific markers to phenotypically profile immune cells across patients. To screen for antigen-specific CD8+ T cells a highly-multiplexed combinatorial tetramer staining using ten different metal-labelled streptavidins was employed (9). 120 possible combinations were generated to screen for 1091 putative neoantigens, 123 tumor-associated antigens (TAA) and 46 cancer-unrelated epitopes (mostly virus-derived).


The detection of neoantigen-specific CD8+ T cells in circulation and in the tumor is challenging. The number of mutations found in some human cancers is rather low, thus reducing the probability that unique, immunogenic neoantigens are expressed. Moreover, limitations of neoantigen candidate prediction algorithms may result in the identification of epitopes that are not presented by the tumor cells and/or not immunogenic. In total, we screened for 1091 putative neoantigens, 123 tumor-associated antigens (TAA) and 46 cancer-unrelated epitopes (mostly virus-derived). Two positive hits were detected for neoantigen epitopes from a total of 24 patients using mass cytometry coupled to MHC-tetramer staining (fig. 1). The small number of neoantigen-specific T cell populations detected may be related to the relatively low mutational burden of these tumors. Interestingly, we detected cancer-unrelated MHC-tetramer+ cells specific for various epitopes derived from Epstein Barr virus (EBV), human cytomegalovirus (HCMV) or influenza virus.

Figure 1. Total number of different MHC class-I tetramers screened for neoantigens, TAA and cancer-unrelated epitopes by mass cytometry (left). Total number of different MHC class-I tetramers identified for neoantigens, TAA and cancer-unrelated epitopes by mass cytometry (right). Neoantigens are colour-coded by patient.

Neoantigen-specific CD8 T cells were detected in two patients (fig. 2a). Interestingly, we detected cancer-unrelated CD8+ TILs in cohorts of patients with lung cancer or colorectal cancer (in  37.5% of lung cancer patients and in 50% of colorectal cancer patients) (fig. 2b). In these cases, CD8+ TILs were specific for various viral epitopes. These data demonstrate that within TILs, CD8+ T cells are not all specific for tumor antigens, but may include bystander CD8+ T cells specific for cancer-unrelated epitopes.

Figure 2. Tumor-specific and cancer-unrelated CD8+ T cells infiltrate tumor tissues.
a) Flow cytometry dot plots representing CD8+ TILs identified from colorectal tumor CD3+ TILs using mass cytometry screening. MHC-tetramer mutated (mut) DST or mutAHR epitope.
b) Representative flow cytometry dot plot showing cancer-unrelated CD8+ TILs specific for different viral epitopes, identified from colorectal tumor CD3+ TILs using mass cytometry screening. Percentages are of MHC tetramer+ cells among CD8+ TILs for each patient.

After screening and identifying cancer-unrelated and tumor-specific CD8+ TILs, we compared the phenotypes of these two populations with one another and the remaining CD8+ TILs of unknown specificity. All the neoantigen-specific CD8+ TILs that we identified displayed resident memory T cell-like phenotypes and expressed various co-stimulatory and inhibitory receptors, such as PD-1 (Fig. 3). We observed diverse, heterogenous  phenotypic profiles for cancer-unrelated CD8+ TILs with respect to this analysis, and most of these markers were similarly expressed as observed on neoantigen-specific CD8+ TILs. Many of the CD8+ TILs also expressed a resident memory T cell-like phenotype as well as various co-stimulatory and activation markers. Interestingly, we observed a striking lack of CD39 expression on cancer-unrelated CD8+ TILs in contrast to high expression by tumor-specific CD8+ TILs. These data indicate that tumors can comprise a wide range of frequencies and phenotypes of tumor infiltrating CD8+ T cells, and that measuring CD39 expression might have utility to identify and discriminate cancer-unrelated from tumor-specific CD8 T cells.


Figure 3. Cancer-unrelated CD8+ TILs do not express CD39. Expression of markers by cancer-unrelated MHC tetramer+ cells (blue) and tumor-specific MHC tetramer+ CD8+ TILs (red) in human tumors.

Furthermore, we analysed peripheral blood from a patient with microsatellite-instable metastatic colorectal cancer who responded rapidly to anti-PD-1 treatment (pembrolizumab). The proliferating CD8+ T cells in the blood from this patient, identified by Ki67 expression, were also characterized by high expression of CD39, indicating that the expansion of this population in the peripheral blood of a patient may be a proxy for response to anti-PD1 treatment.

Figure 4. Schematic for blood collection in colorectal cancer patients treated with pembrolizumab (anti-PD-1 treatment). (left). Flow cytometry dot blot representing co-expression of CD8+ T CD39+and Ki67+ cells (red) among bulk CD8 (gray)(right).

CD39 might play a promising role as a therapeutic target or immune monitoring biomarker during  immune checkpoint therapy. Several groups have independently confirmed our findings utilizing CD39 as a component of their analytical strategy in-house. Duhen et al. reported that CD103+ CD39+ CD8+ TILs showed an anti-tumor effect, and higher frequencies of CD103+CD39+ CD8 TILs in patients with head and neck cancer were associated with better overall survival. Moreover, they proposed that using CD39 and CD103 to enrich for tumor-reactive CD8 T cells prior to TIL expansion might be a strategy to improve the clinical success of adoptive immunotherapy (11). Others have observed that targeting CD39 and CD73 synergistically promotes T cell activation in cancer patients (12). In conclusion, these data support our results that CD39 may be used as a promising marker of tumor-specific CD8+ T cells, and might be utilized as a predictive biomarker of patient treatment response during cancer immunotherapy.


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