T Cell Phenotypes in Upper Gastrointestinal Cancers

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Abstract

AbstractBackground: Upper gastrointestinal adenocarcinomas are increasing in prevalence, particularly oesophageal cancer where the incidence rates have increased by over 65% in the last 30 years. Both adenocarcinomas are associated with very poor outcomes; the 5 year survival rate for oesophageal cancer being only 15% and gastric cancer 19%. There has been limited research into the role of the immune system in the development and treatment of upper gastrointestinal adenocarcinomas. Some studies have suggested that certain T helper subsets, including Th17 and Th22 cells, may play a role in these cancers and could therefore be a potential area of further study. However the classification of these cell types has altered over time in part due to the increased availability of marker–specific antibodies and increased power of multi-colour flow cytometry technology. Thus, the limited ability to clearly detect specific T cell subsets in earlier studies may have contributed to the contradictory roles predicted for these T helper subsets cells in the development of upper gastrointestinal malignancy. Aims: This project focuses on developing multi-colour flow cytometry panels that can accurately identify the diversity of T helper cell phenotypes and use these techniques to further explore the role these cells may have in the development and disease process of upper gastrointestinal adenocarcinomas.Methods: Blood samples from healthy donors and patients with either gastric adenocarcinoma or oesophageal adenocarcinoma would be collected and matched peripheral blood mononuclear cells (PBMC) isolated. Experiments were performed to develop an optimized flow cytometry panel that could identify the subtype populations of CD4 cells Th17 and Th22 by varying the duration of activation and the freeze / thawing of the PBMCs. Alongside this process, a flow cytometry technique was developed to optimize the identification of these populations. Once this methodological process was optimised, healthy donor PBMCs and gastric or oesophageal cancer PBMCs were analysed to determine the relative frequency of Th17 and Th22 cells within the different patient and healthy donor cellular populations. Results: The optimal activation period to identification of Th17 and Th22 cells was 16 hours. Moreover, freeze / thawing had minimal impact on the ability to identify these populations thereby facilitating the batched analysis with improved consistency in sample analysis. A flow cytometry panel of 10 flourochromes was also designed and optimised to more accurately improve identification of Th17 and Th22 by their cytokine profiles CD3+CD4+IL17+IL10-INF?- and CD3+CD4+IL22+IL17-TNF?+ respectively. To also identify the possible plastic nature of Th17 cells, further populations were defined by their cytokine profiles Th17/Treg (CD3+CD4+IL-17+IL-10+INF?-) and Th17/Th1 (CD3+CD4+IL-17+IL-10-INF?+). Analyses of these populations in healthy donor PBMCs found a significant positive correlation between the relative frequency of Th17 and Th22 cells, Th17/Th1 and Th22 cells, and also Th17/Th1 and Th17 cells suggesting a possible relationship between these phenotypes.When comparisons were made between healthy donor PBMCs and patients with gastric cancer or oesophageal cancer, there was an observed increased in the relative frequency of Th17, Th22 and Th17/Th1 cell populations but not Th17/Treg cells in patients with either oesophageal or gastric cancer. There was also evidence of an increase in the populations of Th22, Th17/Th1 and Th17/Treg cells with increasing disease stage. However, the converse was seen in the PBMCs of patients with gastric cancer as the percentages of Th22 and Th17 populations decreased with increasing disease stage suggesting a negative correlation. Conclusion: These results show that the phenotypes Th22 and Th17 were accurately identified using a ten-flourochrome flow cytometry panel in both healthy donors and cancer patients. There was also a relationship between Th17 and Th22 cells in both gastric and oesophageal cancers, however, the role they play maybe very different in the two different types of adenocarcinoma potentially suggesting a different pathophysiological process. Furthermore there was evidence of different subtypes of Th17 populations including Th17/Th1 and Th17/Treg that may play very different roles in the disease process highlighting the plasticity of Th17 cells.

Keyword(s)

T Cells; Upper GI Cancers

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