Transcriptome-wide Mendelian randomization exploring dynamic CD4+ T cell gene expression in colorectal cancer development

Abstract

Recent research suggests higher circulating lymphocyte counts may protect against colorectal cancer (CRC). However, the role of specific lymphocyte subtypes and activation states remain unclear. CD4+ T cells-a highly dynamic lymphocyte subtype-undergo gene expression changes upon activation that are critical to their effector function. Previous studies using bulk tissue have limited our understanding of their role in CRC risk to static associations. We applied Mendelian randomization (MR) and genetic colocalisation to evaluate causal relationships of gene expression on CRC risk across multiple CD4+ T cell subtypes and activation states. Genetic proxies were obtained from single-cell transcriptomic data, allowing us to investigate the causal effect of expression of 1,805 genes across CD4+ T cell activation states on CRC risk (78,473 cases; 107,143 controls). Analyses were stratified by CRC anatomical subsites and sex, with sensitivity analyses assessing whether the observed effect estimates were likely to be CD4+ T cell-specific. We identified 6 genes-FADS2, FHL3, HLA-DRB1, HLA-DRB5, RPL28, and TMEM258-with strong evidence for a causal role in CRC development (FDR-P < 0.05; colocalisation H4 > 0.8). Causal estimates varied by CD4+ T cell subtype, activation state, CRC subsite and sex. However, many of genetic proxies used to instrument gene expression in CD4+ T cells also act as eQTLs in other tissues, highlighting the challenges of using genetic proxies to instrument tissue-specific expression changes. We demonstrate the importance of capturing the dynamic nature of CD4+ T cells in understanding CRC risk, and prioritize genes for further investigation in cancer prevention.

Keywords: CD4+ T cells; Mendelian randomization; colorectal cancer; gene expression; genetic epidemiology.

Fig. 1.

Overview of CD4+ T cell activation states and subtypes A) CD4+ T cells undergo complete remodeling of gene expression to shape their effector function upon activation. This activation occurs in distinct stages, progressing from a resting stage (0 h), to minimally active cells following activation [“lowly active” (LA)], before undergoing cell division (16 h post-activation), after completion of the first cell division (40 h post-activation), and after acquiring effector functions (5d post-activation). Each activation timepoint, or state, reflects a unique functional and transcriptional profile crucial for immune surveillance and response. Color shadings reflect transcriptomic changes. B) Circulating CD4+ T cell subtypes can broadly be grouped into 3 functional clusters: (i) naïve T cells (CD4 naïve)—cells that have not yet encountered an antigen; (ii) memory T cells (CD4 memory)—essential for rapid and robust responses to previously encountered antigens; (iii) regulatory T cells (nTreg)—pivotal for maintaining immune homeostasis by suppressing excessive immune responses.

Fig. 2.

Flow chart describing study methodology. Note that genetic instruments were not available for all cell subtypes at every activation timepoint, meaning the total number of CD4+ T cell expression profiles investigated does not exactly equal the number of timepoints multiplied by the number of cell subtypes. CRC, colorectal cancer; eQTL, expression quantitative trait loci; GTEx, Genotype-Tissue Expression.

Fig. 3.

Volcano plot showing 2-sample MR results. Colors represent the different CD4+ T cell subtypes, shapes represent CRC subsites and sex, and point size represents activation state. Points labeled with gene names. Refer to Table S1 for CD4+ T cell subtype definitions and abbreviations.

Fig. 4.

Proportion of CD4+ T cell activation states and cell subtypes contributing to significant MR results. A) Stacked bar plot representing proportion of CD4+ T cell activation states (0 h, LA, 16 h, 40 h, 5 d) contributing to main study. B) Pie chart representing proportion of CD4+ T cell subtypes contributing to main study results. Refer to Table S1 for CD4+ T cell subtype definitions and abbreviations. LA, lowly active.

Fig. 5.

MR results for genes that showed strong evidence of genetic colocalisation, separated by gene, sex and CRC subsites. Colors represent CD4+ T cell subtypes; shapes represent activation timepoint; and point fill represents whether FDR-P is < or > 0.05. CRC, colorectal cancer; LA, lowly active.