BACKGROUND: Recent research has identified a potential protective effect of higher numbers of circulating lymphocytes on colorectal cancer (CRC) development. However, the importance of different lymphocyte subtypes and activation states in CRC development and the biological pathways driving this relationship remain poorly understood and warrant further investigation. Specifically, CD4+ T cells - a highly dynamic lymphocyte subtype - undergo remodelling upon activation to induce the expression of genes critical for their effector function. Previous studies investigating their role in CRC risk have used bulk tissue, limiting our current understanding of the role of these cells to static, non-dynamic relationships only. METHODS: Here, we combined two genetic epidemiological methods - Mendelian randomisation (MR) and genetic colocalisation - to evaluate evidence for causal relationships of gene expression on CRC risk across multiple CD4+ T cell subtypes and activation stage. Genetic proxies were obtained from single-cell transcriptomic data, allowing us to investigate the causal effect of expression of 1,805 genes across five CD4+ T cell activation states on CRC risk (78,473 cases; 107,143 controls). We repeated analyses stratified by CRC anatomical subsites and sex, and performed a sensitivity analysis to evaluate whether the observed effect estimates were likely to be CD4+ T cell-specific. RESULTS: We identified six genes with evidence (FDR-P<0.05 in MR analyses and H4>0.8 in genetic colocalisation analyses) for a causal role of CD4+ T cell expression in CRC development - FADS2, FHL3, HLA-DRB1, HLA-DRB5, RPL28, and TMEM258. We observed differences in causal estimates of gene expression on CRC risk across different CD4+ T cell subtypes and activation timepoints, as well as CRC anatomical subsites and sex. However, our sensitivity analysis revealed that the 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. CONCLUSIONS: Our study demonstrates the importance of capturing the dynamic nature of CD4+ T cells in understanding disease risk, and prioritises genes for further investigation in cancer prevention research.

Cancer Epidemiology and Prevention Research Unit School of Public Health Imperial College London London United Kingdom

Center for Cancer Research Medical University of Vienna Vienna Austria

Colorectal Oncogenomics Group Department of Clinical Pathology The University of Melbourne Parkville VIC 3010 Australia

Department of Clinical Genetics Karolinska University Hospital Stockholm Sweden

Department of Endocrine and Metabolic Diseases Shanghai Institute of Endocrine and Metabolic Diseases Ruijin Hospital Shanghai Jiaotong University School of Medicine Shanghai China

Department of Epidemiology and Biostatistics School of Public Health Imperial College London UK

Department of Epidemiology Harvard T H Chan School of Public Health Boston Massachusetts USA

Department of Epidemiology University of Washington Seattle Washington USA

Department of Laboratory Medicine and Pathology Mayo Clinic Arizona Scottsdale AZ USA

Department of Molecular Biology of Cancer Institute of Experimental Medicine of the Czech Academy of Sciences Prague Czech Republic

Department of Molecular Medicine and Surgery Karolinska Institutet Stockholm Sweden

Department of Oncologic Pathology Dana Farber Cancer Institute Boston Massachusetts USA

Division of Medical Oncology and Hematology Princess Margaret Cancer Centre Toronto Canada

Genomic Medicine and Family Cancer Clinic Royal Melbourne Hospital Parkville Melbourne VIC 3000 Australia

Institute of Environmental Medicine Karolinska Institutet Stockholm Sweden

MRC Integrative Epidemiology Unit University of Bristol Bristol UK

Nutrition and Metabolism Branch International Agency for Research on Cancer WHO Lyon France

Population Health Sciences Bristol Medical School University of Bristol Bristol UK

Program in MPE Molecular Pathological Epidemiology Department of Pathology Brigham and Women's Hospital Harvard Medical School Boston Massachusetts USA

Public Health Sciences Division Fred Hutchinson Cancer Center Seattle Washington USA

School of Cellular and Molecular Medicine University of Bristol Bristol UK

Shanghai National Clinical Research Center for Metabolic Diseases Key Laboratory for Endocrine and Metabolic Diseases of the National Health Commission Shanghai National Center for Translational Medicine Ruijin Hospital Shanghai Jiaotong University School of Medicine Shanghai China

Translational Health Sciences Bristol Medical School University of Bristol Bristol UK

University of Melbourne Centre for Cancer Research Victorian Comprehensive Cancer Centre Parkville VIC 3010 Australia

University of Southern California Department of Population and Public Health Sciences Los Angeles California USA

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