PMC7503213_01
Identification of EBV-specific CD8+ TILs in EBV-infected LELC of the lung. (a) Immunohistochemistry staining of the lung tumor from LELC patient stained with haematoxylin (blue) and Epstein-Barr virus-encoded small RNA in situ hybridisation (EBERish - brown) (patient A311). (b) Frequency of EBV-specific CD8+ T cells for EBV lytic and EBV latent epitopes in HLA-A*24: 02 LELC patient (n = 1) and healthy individuals (n = 7). (c) Flow staining of sorted EBV (BMLF1)-specific CD8+ TILs using HLA-A*24: 02 tetramer (DYNFVKQLF) (left panel). Frequency of tetramer-positive cells among CD8+ TILs. TCRalpha and TCRbeta clones' repertoire of bulk EBV(BMLF1)-specific CD8+ TILs (right panel). Data from LELC patient A311. (d) Flow staining of sorted EBV (BRLF1)-specific CD8+ TILs using HLA-A*24: 02 tetramer (TYPVLEEMF) (left panel). Frequency of tetramer-positive cells among CD8+ TILs. TCRalpha and TCRbeta clones' repertoire of bulk EBV (BRLF1)-specific CD8+ TILs (right panel), * the same CDR3 amino acid sequence but different nucleotide sequence. Data from LELC patient A311. (e) Enrichment of the gene set for exhausted T cells in naive CD8+ T cells from PBMC (flow-sorted CCR7- CD45RO-), and EBV (BMLF1)- or EBV (BRLF1)-specific CD8+ TILs. Gene position on the left indicates enrichment in EBV (BMLF1)- or EBV (BRLF1)-specific CD8+ TILs. Gene position on the right indicates enrichment in naive CD8+ T cells. Data from LELC patient A311. (f) Heat map of gene set for exhaustion shown in e. Data from LELC patient A311.
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We confirmed the presence of EBV viral RNA in the tumor cells of this patient (Figure 1a). Based on the HLA-A alleles expressed by this patient, we screened for EBV-specific CD8+ T cells using HLA-A*24: 02 tetramers loaded with different EBV epitopes (Supplementary figure 1a). Supplementary figure 1a). Since EBV proteins are presented by MHC class I molecules from the tumor cells, EBV-specific CD8+ TILs can be considered as tumor-specific. We did not detect any CD8+ T cells specific for antigens associated with EBV latent cycle in this patient despite the addition of epitopes derived from three latency-associated gene products (LMP2, EBNA3A or EBNA3B) (Figure 1b, see Discussion). We first studied the TCR repertoire and observed that both populations of EBV-specific CD8+ TILs were polyclonal for their TCRalpha with three and four different TRA clones detected for each epitope (Figure 1c and d). Interestingly, BMLF1 tetramer+ cells expressed only one TCRbeta chain B clone (Figure 1c). TRB clones were detected at a similar frequency (Figure 1d). This result highlighted the TCR polyclonality of antigen-specific CD8+ TILs. Gene set enrichment analysis revealed a significant exhausted profile for both populations of EBV-specific CD8+ TILs (Figure 1e). MKI67), we surprisingly observed a dichotomy for some other genes (Figure 1f). For example, the inhibitory receptor PDCD1 (PD-1) was only detected in EBV(BRLF1)-specific CD8+ TILs at low level (Figure 1f). Furthermore, the intensity of PD-1 expression on these cells was lower than that of PD-1 expression on tumor-associated antigen or neoantigen-specific CD8+ TILs (Supplementary figure 1b).
EBV-specific CD8+ TILs in EBV-infected LELC are heterogeneous and partially lack expression of PD-1. (a) UMAP plots of CD8+ T cells from PBMC (left) and tumor tissue (right) isolated from LELC patient A311 and acquired by mass cytometry. (b) Concatenated peripheral and tumor-infiltrating CD8+ T-cell UMAP plot showing expression intensities for various phenotypic surface markers. (c) UMAP plots of EBV (BRLF1)-specific CD8+ T cells (green) plotted on total CD8+ T cells (grey) in PBMC and tumor from LELC cancer patient (left panel). Based on the clusters identified for BRLF1-specific CD8+ T cells, frequencies of cells positive for each surface marker were calculated and shown in a heat map (right panel). (d) UMAP plots of EBV (BMLF1)-specific CD8+ T cells (blue) plotted on total CD8+ T cells (grey) in PBMC and tumor from LELC cancer patient (left panel). Based on the clusters identified for BMLF1-specific CD8+ T cells, frequencies of cells positive for each surface marker were calculated and shown in a heat map (right panel). (e) Flow-cytometry staining showing expression of PD-1 by EBV (BMLF1)- and EBV (BRFL1)-specific CD8+ TILs. Frequencies among tetramer+ CD8+ TILs. (f) Immunohistochemistry staining of the lung tumor from LELC patient stained with haematoxylin (blue) and PD-L1 (brown) (patient A311).
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For high-dimensional analysis of tetramer+ CD8+ T cells, we used uniform manifold approximation and projection (UMAP), which accounts for non-linear relationships between markers and projects high-dimensional data into two dimensions (called UMAP1 and UMAP2) by making a pairwise comparison of cellular phenotypes to optimally plot similar cells nearby to each other. 21 ,  22  We observed that CD8+ T cells derived from PBMC or tumor were localised in different areas of the UMAP plot, indicating non-overlapping phenotypes of CD8+ T cells between the two tissues (Figure 2a). In PBMC, we observed clusters of CD8+ T cells that were identified with characteristics of naive (CCR7+ CD45RO-), effector (CCR7- CD45RO+) or senescent (CD57+) cells (Figure 2a, b). In the tumor tissue, CD8+ TIL clusters expressed tissue-resident memory (Trm markers) (CD69+ CD103+/-) (Figure 2b). When plotting both populations of EBV-specific CD8+ T cells on the UMAP plot, we observed the presence of two different clusters in PBMC (clusters 1 and 2) and four in tumor tissue (clusters 3 to 6), highlighting their phenotypic heterogeneity (Figure 2c, d). TILs expressed CD39 - a marker reported to be induced after chronic antigen stimulation - but not PD-1 (Figure 2c, d). The heterogeneity of both EBV-specific CD8+ TILs was mainly driven by differential expression of integrin CD103 (cluster 3), the senescence marker CD57 (cluster 4) or the absence of adhesion molecule CD56 (cluster 5). Surprisingly, the two populations of EBV-specific CD8+ TILs infiltrating this EBV-driven LELC tumor only partially expressed PD-1, 13.8% and 52.6% of tetramer+ cells, respectively, for BMLF1 and BRLF1 epitopes (Figure 2e). Nonetheless, we noted that the tumor cells expressed high level of PD-L1 in this tumor (Figure 2f).