Increased circulating regulatory T cells and decreased follicular T helper cells are associated with colorectal carcinogenesis

Abstract

Objective: Colorectal cancer (CRC) is the third most prevalent cancer worldwide and is associated with high morbidity and mortality rates. Colorectal carcinogenesis occurs via the conventional adenoma-to-carcinoma and serrated pathways. Conventional T helper (Th) and innate lymphoid cells (ILCs) play vital roles in maintaining intestinal homeostasis. However, the contribution of these two major lymphoid cell populations and their associated cytokines to CRC development is unclear. Therefore, we aimed to analyze peripheral lymphocyte profiles during colorectal carcinogenesis.

Methods: We collected 86 blood samples concurrently, and pathologists confirmed the presence of various pathological conditions (i.e., HPs, adenoma, and carcinoma) using hematoxylin and eosin staining. Ten healthy donors were recruited as healthy controls (HCs) from the physical examination center. We performed flow cytometry on peripheral blood mononuclear cells collected from patients with various pathological conditions and the HCs, and cytokines (interleukin-2, interleukin-4, interleukin-5, interleukin-13, interleukin-17A, interleukin-17F, interleukin-22, interferon-γ, and tumor necrosis factor-α) were quantified. We also analyzed the published single-cell RNA sequence data derived from tissue samples from different stages of colorectal carcinogenesis.

Results: The cytokine response in peripheral CD4⁺ T cells was upregulated during the carcinoma process. The frequency of peripheral regulatory T cells (Tregs) increased in the adenoma and carcinoma stages. While the T follicular helper (Tfh) cell proportion was downregulated in the adenoma and carcinoma processes. Thus, Th cell subsets, especially Tregs and Tfh cells, were involved in colonic diseases. Moreover, the immunological profile characteristics in the HPs were clarified.

Conclusion: We comprehensively analyzed circulating ILCs and adaptive T-cell lymphocyte subtypes in colorectal carcinoma progression. Our results show the immunological profile characteristics and support the involvement of Th subsets, especially Treg and Tfh cell populations, in colonic diseases. These findings significantly enhance our understanding of the immune mechanisms underlying CRC and its precancerous lesions. Further investigation of the Treg and Tfh cells' function in colorectal disease development will provide potential therapeutic targets for monitoring and preventing CRC development.

Keywords: T helper cell; adenoma; colorectal cancer; hyperplastic polyps; innate lymphoid cells.

Figure 1

Study flowchart and pathologic diagnosis of enrolled patients. (A) Subdivision of enrolled subjects into health controls, hyperplastic polyps, adenoma (grades I, II, and III), and carcinoma. The peripheral blood samples were collected, and the key lymphocyte subpopulations in PBMCs and the plasma cytokines were analyzed. (B) Representative H&E-stained sections of a cascade of colonic lesions. Scale bar: 500 μm (×100 magnification).

Figure 2

Plasma cytokine analysis during a cascade of colonic lesions. (A) The concentration of TNF-α, IFN-γ, and IL-2 in the plasma. (B) The concentration of IL-5, IL-13, and IL-4 in the plasma. (C) The concentration of IL-17A, 17F, and 22 in the plasma. Each dot represents one donor. HC, healthy control. Error bars represent the SEM. ANOVA or non-parametric test (Kruskal–Wallis test) as appropriate. All results are not significantly different. n = 10–19 in each group.

Figure 3

Th cell response analysis during a cascade of colonic lesions. (A–C) Flow cytometry quantification of the frequency of CXCR3⁺ (A), CCR4⁺ (B), and CCR6⁺ (C) cells in CD4⁺ T cells during a cascade of colonic lesions. (D-F) Flow cytometry quantification of the frequency of IFN-gamma⁺ (D), IL-4⁺ (E), and IL-17A⁺ (F) cells in CD4⁺ T cells during a cascade of colonic lesions. Each dot represents one donor. HC, healthy control. Error bars represent the SEM. *P < 0.05; **P < 0.01 (ANOVA or non-parametric test as appropriate). n = 10–19 in each group.

Figure 4

The change of ILC cell populations during a cascade of colonic lesions. (A–C) Flow cytometry quantification of the frequency of Lin^-CD127⁺CRTH2^-CD117^- ILC1 cells (A), Lin^-CD127⁺CRTH2⁺CD117^- ILC2 cells (B), and Lin^-CD127⁺CRTH2^-CD117⁺ ILC3 cells (C) in CD45⁺ PBMCs during a cascade of colonic lesions. (D–F) Flow cytometry quantification of the frequency of Lin⁻CD127⁺IFN-gamma⁺ cells (D), Lin⁻CD127⁺IL-5⁺ cells (E), and Lin⁻CD127⁺IL-22⁺ cells (F) in CD45⁺ PBMCs during a cascade of colonic lesions. Each dot represents one donor. HC, healthy control. Error bars represent the SEM. *P < 0.05; **P < 0.01 (ANOVA or non-parametric test as appropriate). n = 10–19 in each group.

Figure 5

Treg cell analysis during a cascade of colonic lesions. (A) The representative flow plots about the gating strategy of CD25⁺ Treg cells in CD4⁺ T cells during a cascade of colonic lesions. (B) Flow cytometry quantification of the frequency of CD4⁺CD25⁺ Treg cells in CD4⁺ T cells. Each dot represents one donor. HC, healthy control. Error bars represent the SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (ANOVA or non-parametric test as appropriate). n = 10–19 in each group. (C) IL-2RA (CD25) and FOXP3 expression analysis in the tissue-derived CD4⁺ T cells among different colonic lesions by analyzing the single-cell RNA-sequencing data (GSE161277). The color depth represents the average expression, and the size of the dots represents the percentage expressed.

Figure 6

The change of Tfh cells during a cascade of colonic lesions. (A) The FACS staining (left) and frequency quantification (right) analysis of CD4⁺CXCR5⁺ PD1⁺ cells in CD4⁺ T cells during a cascade of colonic lesions. (B) The expression analysis of PDCD1 and CXCR5 in the tissue-derived CD4⁺ T cells among different colonic lesions by analyzing the single-cell RNA-sequencing data (GSE161277). The color depth represents the average expression, and the size of the dots represents the percentage expressed. (C) The FACS staining (left) and frequency quantification (right) analysis of CD19⁺CD38⁺CD27⁺ plasma cells during a cascade of colonic lesions. (D) The FACS staining (left) and frequency quantification (right) analysis of IL-21⁺ cells in CD4⁺ T cells during a cascade of colonic lesions. Each dot represents one donor. HC, healthy control. Error bars represent the SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (ANOVA or non-parametric test as appropriate). n = 10–19 in each group.