Krisna, S. S. | Search bioRxiv

bioRxiv2024

Immunometabolic analysis of primary murine Group 2 Innate Lymphoid Cells: a robust step-by-step approach

Group 2 Innate Lymphoid Cells (ILC2s) have recently been shown to exert key regulatory functions in innate and adaptive immune response networks that drive the establishment and progression of type 2 immunity and its associated pathologies. Although mainly tissue resident, ILC2s and their crosstalk within tissue microenvironments influences both local and systemic metabolism. In turn, the metabolic status shapes the diverse ILC2 phenotypes and effector functions. Hence, deciphering the metabolic networks of ILC2s is essential in understanding ILC2s roles in health as well as pathophysiologies. Here we detail a framework of experimental approaches to study key immunometabolic states of primary murine ILC2s and link them to phenotypes and functionality. Utilizing flow cytometry, Single Cell ENergetIc metabolism by ProfilIng Translation inhibition (SCENITH) as well as the Seahorse platform we provide a framework that allows in-depth analysis of cellular bioenergetic states to determine the immunometabolic wiring of ILC2. Linking immunometabolic states and networks to ILC2 phenotypes and effector functions will allow in-depth studies that assess the potential of novel pharmaceutics to alter ILC2 functionality in experimental and clinical settings.

immunology↗

bioRxiv2020

The pleiotropic effects of the MNK1/2-eIF4E axis support immune suppression and metastasis in a model of postpartum breast cancer.

PurposeBreast cancer diagnosed within 10 years following childbirth is defined as postpartum breast cancer (PPBC) and is highly metastatic. Interactions between immune cells and other stromal cells within the involuting mammary gland are fundamental in facilitating an aggressive tumor phenotype. The MNK1/2-eIF4E axis promotes the translation of pro-metastatic mRNAs in tumor cells, but its role in modulating the function of non-tumor cells in the PPBC microenvironment, and in particular its activity in human PPBC, has not been explored. Experimental designWe used a combination of in vivo PPBC models and in vitro assays to study the effects of phospho-eIF4E deficiency on the pro-tumor function of select cells of the TME. Furthermore, we employed Imaging Mass Cytometry on PPBC and non-PPBC patient samples, to chart the expression of the MNK1/2-eIF4E axis components in the TME. ResultsHere, we show that phospho-eIF4E deficient (eIF4ES209A) PPBC mice are protected against lung metastasis and reveal differences in the lung immune microenvironment of the WT and eIF4ES209A mice. Moreover, we show that the expression of fibroblast-derived IL-33, an alarmin known to induce invasion, was repressed upon MNK1/2-eIF4E axis inhibition. Imaging Mass Cytometry results indicated that human PPBC contain phospho-eIF4E high-expressing tumor cells and CD8+ T cells displaying an activated dysfunctional phenotype. Finally, we block lung metastasis in PPBC mice, using combined MNK1/2 inhibition and anti-PD-1 therapy. ConclusionsThese findings implicate the involvement of the MNK1/2-eIF4E axis during PPBC metastasis and suggest a promising immunomodulatory route to enhance the efficacy of immunotherapy by blocking phospho-eIF4E. Translational relevancePostpartum breast cancer (PPBC) is highly aggressive. It is hypothesized that involution-induced changes in the postpartum breast microenvironment, which include an influx of inflammatory immune cells and activation of resident fibroblasts, facilitate the invasiveness of an existing neoplasm. We used imaging mass cytometry to do an in-depth profiling of the MNK1-eIF4E axis in the TME of a unique cohort of PPBC and non-PPBC patients. We observed patterns of phospho-eIF4E in non-tumor cells that were specific to the TME of PPBC. We also noted that the CD8+ T cells present in PPBC express an activated dysfunctional phenotype characterized by the co-expression of HLA-DR and PD-1. This study represents a first look at the expression of the MNK1-eIF4E axis in the stromal cells of metastatic breast cancer and has therapeutic implications as we show, in an animal model of PPBC, that MNK1/2 inhibition can be used to sensitize tumors to anti-PD1 immunotherapy.

cancer biology↗