The Expression And Clinical Signification Of Follicular Helper T Cells And Follicular Regulatory T Cells In Peripheral Blood Of Colorectal Cancer Patients

Background: Colorectal cancer is the third most commonly diagnosed cancer in the world, over one million colorectal cases are clinically diagnosed each year. Furthermore, the effects of traditoinal treatments( such as operation, radiotherapy, chemotherapy, targeted therapy) are not ideal. Besides the mechanism of the tumor itself, the tumor microenvironment, especially the inflammatory and immune response are related to tumor progression or even radiotherapy, chemotherapy and targeted drug resistance. Thus, the age of cancer immunotherapy comes. In fact, increasing evidence indicate that the immune system plays a critical role in preventing the genesis, growth and metastasis of tumor.The study on CD4+ T lymphocyte subsets is a focus of immunology research in recent years. Follicular helper T cells(Tfh) which express CXCR5 are recently identified as CD4+ T helper lineage. By the chemotaxis of CXCL13, Tfh is recruited to the lymphoid follicle to participate in inducting B cells differentiation and the formation of germinal center. Through the production of cytokines such as IL-21 and adhesion molecules like ICOS to regulate the body’s immune function. Tfh cells’ differentiation, development or related molecular abnormalities will lead to immune dysfunction. As regulation counterparts of Tfh cells,these follicular regulatory T cells(Tfr) share phenotypic characteristics with Tfh and conventional Foxp3+ regulatory T cells(Treg), which inhibit the formation of germinal center and B cells’ differentiation, acting as an opposite role compared with Tfh. Recent studies suggest that dysregulation of immune system plays a critical role in developing breast cancer, non-small cell lung cancer and osteosarcoma. However, little is know about the frequency of Tfh and Tfr expression in colorectal cancer patients(CRC). In this study, we investigated the proportion of Tfh and Tfr, as well as the concentration of IL-21 in serum from peripheral blood of colorectal cancer patients and healthy people by flow cytometry and enzyme-linked immunosorbent assay, and examined the potential association of these cells with different clinicopathological parameter, then analyzing the potential significance of these cells in colorectal cancer.Objective: To investigate the expression of Tfh and Tfr from peripheral blood of colorectal cancer patients and healthy people, as well as the concentration of IL-21 in serum, We examined the potential association of these cells with different clinicopathological parameter, then analyzing the potential significance of these cells in colorectal cancer.Methods:1 By flow cytometry, we quantified the CD4+CXCR5+PD-1+Foxp3- Tfh cells and CD4+CXCR5+PD-1+Foxp3+ Tfr cells from peripheral blood of the experimental group of CRC patients and the control group of healthy people.2 Measuring the concentration of IL-21 at the molecular level in serum from peripheral blood of the experimental group of CRC patients and the control group of healthy people by using enzyme-linked immunosorbent assay method.Results:1 The demographic and clinicopathological parameter of subjectDemographic and other selected characteristics of the cases and controls are shown in Table 1. The experimental group had 62 cases, in which 34 cases were male and 28 cases were female, ranging from 23 to 83 years old, and the mean age was 56.7 years old, the median age was 58.5 years old, the pathology of them was adenocarcinoma, whose TNM stage was: 7 cases of stageⅠ, 21 cases of stageⅡ, 25 cases of stage Ⅲ, 9 cases of stage Ⅳ(four cases of hepatic metastases,three cases of pulmonary metastases,one case of abdominal cavity metastases and one case of brain metastases). These patients of Ⅰ, Ⅱ, Ⅲ and some Ⅳ stage(4 patients with liver metastases) recevied surgery.All of patients with lymph node metastasis were 32 cases and 30 cases without.The control group of healthy people had 30 cases, in which 6 cases were male and 24 cases were female, ranging from 26 to 78 years old, and the mean age was 53.2 years old, the median age was 54 years old. We compared CRC patients with the control group of healthy people on the aspects of age, sex, smoking status, etc., thus there was no statistically differences.2 We quantified the CD4+CXCR5+PD-1+Foxp3-Tfh cells and CD4+CXCR5+PD-1+Foxp3+ Tfr cells from peripheral blood of the CRC patients and the healthy people through flow cytometry. As shown in Fig.1: P1 gate is peripheral blood lymphocytes, and we set CD4+ T cells as the 2th gate, Q3 as CD4+CXCR5+PD-1+Foxp3-Tfh cells, and Q4 as CD4+CXCR5+PD-1+Foxp3+ Tfr cells.3 As shown in Fig.2, we can see an upregulated level of circulating Tfh cells in CD4+ T cells in CRC patients than in control group(2.93±2.48vs2.09±1.34, P=0.038); while the percentage of Tfr cells in CD4+ T cells in CRC patients are less than healthy control group(0.16±0.09 vs 0.2±0.11, P=0.047).4 Analyzing the correlation between peripheral blood Tfh cells and clinicopathological parameter in CRC patients(1) The percentage of Tfh cells in CD4+ T cells with lymph node metastasiswas higher is than without(2.85±1.5 vs 2.20±1.2).(2) Comparing the percentage of Tfh cells in CD4+ T cells between each TNM staging: stageⅠ(1.39±0.64) vs stage Ⅳ(1.97±0.82), which P value was 0.04, thus it had statistically differences; but the p value was all greater than 0.05 by comparing stageⅠwith stageⅡor stage Ⅲ, as well as comparing stageⅡ, Ⅲ, Ⅳ to each other, thus there was no statistically differences.(3) Dividing the experimental group of CRC patients into two groups by the condition of CEA>5ng/ml or CEA<5ng/ml, and comparing the percentage of Tfh cells in CD4+ T cells,(3.06±2.79 vs 2.19±2.16,P=0.173), thus there was no statistically differences.5 Measuring the level of IL-21 in serum of CRC patients and the control group of healthy people by using enzyme-linked immunosorbent assay method,and the results showed that the IL-21 level in the CRC patients was higher than control group.(196.48±69.85 vs 149.85±91.49,P<0.05).Conclusions:1 The expression of Tfh cells is increasing in peripheral blood of CRC patients, accompanied by the decreased expression of Tfr, as well as an increasing level of IL-21 in serum.2 This study suggest that Tfh and Tfr cells may participate the progress of colorectal cancer.