Transcriptomic features of tumour-infiltrating CD4lowCD8high double positive αβ T cells in melanoma

Abstract

Peripheral CD4⁺CD8⁺ double positive (DP) T cells are a phenotypically and functionally heterogeneous population depending on their origin and pathologic context. We previously identified among tumour infiltrating lymphocytes in melanoma, a tumour-reactive MHC class-I restricted CD4^lowCD8^high DP αβ T-cell subpopulation with CD4-like function. In this study, we used an in-depth comparative transriptomic analysis of intra-melanoma DP T cells and CD4 and CD8 single positive (SP) T cells, to better comprehend the origin of this DP phenotype, and define the transcriptomic signature of activated DP T cells. We observed that intra-melanoma DP T cells were transcriptome-wise closer to their CD8 SP T-cell counterparts in terms of number of genes differentially expressed (97 in common with CD8 SP T cells and 15 with CD4 SP T cells) but presented hallmarks of a transition to a CD4-like functional profile (CD40LG) with a decreased cytotoxic signature (KLRC1) in favour of an increased cytokine-receptor interaction signature (IL4, IL24, IL17A…). This unleashed CD4-like program could be the results of the observed unbalanced expression of the THPOK/Runx3 transcription factors in DP T cells. Overall, this study allow us to speculate that intra-melanoma DP T cells arise from CD8 SP T cells being reprogrammed to a helper function.

Figure 1

TCR Vβ repertoire distribution between FACS-sorted and expanded intra-melanoma DP, CD4 SP and CD8 SP T cells from TILs patients. Pie charts representing the frequency of expression of each Vβ segment determined by flow cytometry using a panel of 24 anti-Vβ antibodies. The total percentage indicates the overall repertoire covered by these antibodies.

Figure 2

Differentially expressed genes (DEGs) between activated DP and SP CD4 and CD8 T cells. Heatmap and unsupervised hierarchical clustering of the most significantly DEGs between activated DP and (A) CD4 SP T cells or (B) CD8 SP T cells, matching the following criteria: p < 0.05 by analysis of variance, −1.5 ≤ mean log2 fold change ≥ 1.5. A color bar with scales for each heatmap is included, from dark red to dark blue indicative of high and low normalized expression value respectively. This figure was created using the heatmap.2 package implemented in gplots v3.0.3 in R (https://www.rdocumentation.org/packages/gplots/versions/3.0.3/topics/heatmap.2).

Figure 3

Comparative expression profile of genes encoding transcription factors in intra-melanoma DP. Data were normalized with RPLP0 and PPIA expression using ΔΔCt method and presented as the mean log2 fold ratio difference between DP and CD4 or CD8 SP T cells. The error bars are the standard deviation calculated for mean log2 fold change.