Elucidating the influence of the KrasG12D mutation on intestinal immunity beyond cancer

Details

Supervisors

Julie Stockis

Faculty

Faculté de pharmacie et des sciences biomédicales

Degree label

Master [120] en sciences biomédicales, à finalité approfondie

Abstract

Recent research has identified clones carrying somatic mutations in healthy human tissues. Among these mutations are mutations in the proto-oncogene KRAS, whose contribution to the pathogenesis of cancer is well-established. Surprisingly, KRAS mutations were described in the human small intestine (SI), a tissue where the incidence of cancer is extremely low. Knowing the crucial function of intestinal epithelial cells (IECs) in the regulation of mucosal immunity, our laboratory seeks to determine whether KRAS mutations could influence the SI immune niche and thereby modulate intestinal immune responses beyond cancer. To answer to this question, preliminary experiments were performed in Cambridge, UK, using mice expressing the KrasG12D mutation specifically in adult IECs (KrasG12D-IEC mice). Notably, results showed striking changes in the abundance of regulatory T cell (Treg) subsets in the SI lamina propria (SI LP) of KrasG12D-IEC mice compared to littermate controls. Hence, the objectives of my master thesis were: 1/ to confirm the changes seen in the Treg population in newly imported KrasG12D-IEC mice; 2/ to investigate the mechanisms underlying these changes; 3/ to assess the impact of these modifications on dietary antigen tolerance, given the important immunosuppressive role played by Tregs in this process. To this end, we first developed an 18-colour full spectrum flow cytometry panel and confirmed a decrease in the percentage of RORγt+ Tregs in KrasG12D-IEC mice, yet absolute numbers remained unchanged. On the other hand, we observed an increase in both RORγt- thymic-derived Tregs (tTregs) percentage and numbers in these animals. We further developed an imaging method to confirm these results on paraffin-embedded sections and visualize the localization of these Treg subsets in the SI LP, although it did not prove sensitive enough. In addition, we observed a decrease in MHC- II expression by KrasG12D-expressing IECs, but it remains unclear whether this plays any role in the modulation of the Treg compartment observed. Finally, we established a mouse model of food allergy in susceptible BALB/c mice to monitor the immune response upon antigenic challenge, in terms of anti-allergen serum immunoglobulin E (IgE) as well as immune cell activation in the SI. Unfortunately, we were not able to reproduce such a response in C57bl/6 mice, irrespective of the presence of the KrasG12D mutation. Taken together, we have demonstrated that the presence of the KrasG12D mutation in non-neoplastic intestinal epithelium does have an influence on the intestinal immune niche but its impact on the tolerance to food antigens remains to be determined.