Elucidating the mechanism of type 2 innate lymphoid cell-mediated tumour elimination

Besides amplifying type 2 immune responses, the role of type 2 innate lymphoid cells (ILC2s) in cancer remains ambiguous. While some studies suggest that ILC2s exert immunosuppressive functions, others present evidence they enhance anti-cancer immunity. However, their successful isolation from mammal tissues and in vitro expansion continues to pose challenges. This is partly due to their scarcity compared to other leukocyte populations, but also because our current knowledge on ILC2 biology is incomplete. In this study, we focused on ST2+ IL-25Rlo lung resident ILC2s and demonstrate for the first time how mouse type 2 innate lymphoid cells can be cultured, grown and expanded in serum-free media containing interleukin-33 (IL-33), TSLP, IL-2 and IL-7. Moreover, we report that ILC2s from the lungs of mice with primary tumours are able to cross-present the ovalbumin peptide OVA257-264 and effectively cross-prime a population of OT-I cells. Our data also demonstrates that ILC2s from naïve mice are less efficient at antigen cross-presentation, suggesting a plastic cellular identity in the presence or absence of a tumour. To further our understanding of ILC2s harvested from mice with primary tumours, our single-cell RNA sequencing analyses reveal a highly heterogeneous population with several subsets. One of them downregulates markers associated to type 2 identity such as GATA-3, IL-5 or IL-13, but also begins to express pro-inflammatory markers like granzyme B, IFNγ or interferon-induced membrane proteins. We hypothesize that systemic inflammation or a tumour environment can trigger differentiation of a portion of the lung resident ILC2 population into a pro-inflammatory subset. The existence of these cells challenges current paradigms of ILC2 biology and implies the participation of a distinct agent in the process of cancer immune surveillance.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat

Serum free culture for the expansion and study of type 2 innate lymphoid cells

Abstract Type 2 innate lymphoid cells (ILC2s) were discovered approximately ten years ago and their clinical relevance is gaining greater importance. However, their successful isolation from mammalian tissues and in vitro culture and expansion continues to pose challenges. This is partly due to their scarcity compared to other leukocyte populations, but also because our current knowledge of ILC2 biology is incomplete. This study is focused on ST2+ IL-25Rlo lung resident ILC2s and demonstrate for the first time a methodology allowing mouse type 2 innate lymphoid cells to be cultured, and their numbers expanded in serum-free medium supplemented with Interleukins IL-33, IL-2, IL-7 and TSLP. The procedures described methods to isolate ILC2s and support their growth for up to a week while maintaining their phenotype. During this time, they significantly expand from low to high cell concentrations. Furthermore, for the first time, sub-cultures of primary ILC2 purifications in larger 24- and 6-well plates were undertaken in order to compare their growth in other media. In culture, ILC2s had doubling times of 21 h, a growth rate of 0.032 h−1 and could be sub-cultured in early or late phases of exponential growth. These studies form the basis for expanding ILC2 populations that will facilitate the study and potential applications of these rare cells under defined, serum-free conditions