Characterization of circulating T-, NK-, and NKT cell subsets in patients with colorectal cancer: the peripheral blood immune cell profile

Abstract

Objective: As the development and progression of colorectal cancer (CRC) are known to be affected by the immune system, cell subsets such as T cells, natural killer (NK) cells, and natural killer T (NKT) cells are considered interesting targets for immunotherapy and clinical biomarker research. Until now, the role of systemic immune profiles in tumor progression remains unclear. In this study, we aimed to characterize the immunophenotype of circulating T cells, NK cells, and NKT-like cells in patients with CRC, and to subsequently correlate these immunophenotypes to clinical follow-up data.

Methods: Using multiparameter flow cytometry, the subset distribution and immunophenotype of T cells (CD3⁺CD56^-), CD56^dim NK cells (CD3^-CD56^dim), CD56^bright NK cells (CD3^-CD56^bright), and NKT-like (CD3⁺CD56⁺) cells were investigated in peripheral blood mononuclear cell (PBMC) samples from 71 CRC patients and 19 healthy donors.

Results: CRC patients showed profound differences in immune cell subset distribution and their immunophenotype compared to healthy donors, as characterized by increased percentage of regulatory T cells, and reduced expression level of the natural cytotoxicity receptors NKp44 and NKp46 on both CD56^dim NK cells and NKT-like cells. Finally, we showed in a multivariate analysis that above-median percentage of CD16⁺ NKT-like cells was independently associated with shorter disease-free survival in CRC patients.

Conclusion: The altered phenotype of circulating immune cell subsets in CRC and its association with clinical outcome highlight the potential use of PBMC subsets as prognostic biomarkers in CRC, thereby contributing to better insight into the role of systemic immune profiles in tumor progression.

Keywords: Cancer immunology; Colorectal cancer; Natural cytotoxicity receptors; Peripheral blood immune cell profile; Prognostic biomarkers; Tumor progression.

Fig. 1

The peripheral blood immune cell subset distribution in CRC patients compared to healthy donors. The distribution of circulating immune cell subsets was compared between CRC patients (grey dots) and healthy donors (black dots). a The total percentage of T cells (% CD3⁺CD56⁻ cells of total lymphocytes). b Percentage of CD8⁺ T cells (% of total T cells). c Percentage of CD4⁺ T cells (% of total T cells). d Percentage of T_reg (% of CD3⁺CD4⁺ T cells). e The total percentage of NK cells (% CD3⁻CD56⁺ cells of total lymphocytes). f Percentage of CD56^dim NK cells (% of total NK cells). g Percentage of CD56^bright NK cells (% of total NK cells). h The total percentage of NKT-like cells (% CD3⁺CD56⁺ cells of total lymphocytes). The bars show median percentage of the respective immune cell subset including 95% CI

Fig. 2

The peripheral blood immunophenotype of CD56^dim NK cells and NKT-like cells in CRC patients compared to healthy donors. The peripheral blood immune cell profile was compared between CRC patients (grey dots) and healthy donors (black dots). a The percentage of NKp44⁺ CD56^dim NK cells. b Percentage of NKp44⁺ NKT-like cells. c Correlation between the percentage of NKp44⁺ CD56^dim NK cells and NKT-like cells. d Expression level of NKp44 on CD56^dim NK cells. e Expression level of NKp44 on NKT-like cells. f Correlation between expression level of NKp44 on CD56^dim NK cells and NKT-like cells. g Percentage of NKp46⁺ CD56^dim NK cells. h Percentage of NKp46⁺ NKT-like cells. i Correlation between the percentage of NKp46⁺ CD56^dim NK cells and NKT-like cells. j Expression level of NKp46 on CD56^dim NK cells k Expression level of NKp46 on NKT-like cells. l Correlation between expression level of NKp46 on CD56^dim NK cells and NKT-like cells. The bars show median percentage or expression level of the respective immunophenotypic marker including 95% CI

Fig. 3

Relationship between the immune subset distribution and immunophenotype of circulating lymphocyte subsets with DFS of CRC patients. DFS curves are shown for stage II and III CRC patients (N = 49) at risk for development of metastases. Stratifications were based on the median percentage of positive cells or expression level of the respective immunophenotypic marker. a The percentage of T_reg (% CD3⁺CD4⁺ T cells). b NKp30 expression level on CD56^bright NK cells. c Percentage of CD16⁺ circulating NKT-like cells and, d Percentage of CD158a⁺ NKT-like cells. ^*This marker was only studied in 25 CRC patients at risk for development of metastasis (stage II and III)