Liang, H.-E. | Search bioRxiv

bioRxiv2021

Alveolar macrophages strictly rely on GM-CSF from alveolar epithelial type 2 cells before and after birth

Programs defining tissue-resident macrophage identity depend on local environmental cues. For alveolar macrophages (AMs), these signals are provided by immune and non-immune cells, and include GM-CSF (CSF2). However, evidence to functionally link components of this intercellular crosstalk remains scarce. We thus developed new transgenic mice to profile pulmonary GM-CSF expression, which we detected in both immune cells, including group 2 innate lymphoid cells and {gamma}{delta} T cells, as well as AT2s. AMs were unaffected by constitutive deletion of hematopoietic Csf2 and basophil depletion. Instead, AT2 lineage-specific constitutive and inducible Csf2 deletion revealed the non-redundant function of AT2-derived GM-CSF in instructing AM fate, establishing the postnatal AM compartment, and maintaining AMs in adult lungs. This AT2-AM relationship begins during embryogenesis, where nascent AT2s timely induce GM-CSF expression to support the proliferation and differentiation of fetal monocytes contemporaneously seeding the tissue, and persists into adulthood, when epithelial GM-CSF remains restricted to AT2s.

immunology↗

bioRxiv2021

Type 2 innate immunity regulates hair follicle homeostasis to control Demodex pathosymbionts

Allergic skin diseases are common, but basal roles for type 2 immunity in cutaneous homeostasis are incompletely understood. Here, we show that skin group 2 innate lymphoid cells (ILC2s) are the predominant resident cells that secretes IL-13, which attenuates epithelial cell proliferation during anagen throughout the hair follicle (HF), including in HF stem cells. Although HF are normal in the absence of type 2 immunity, colonization with the commensal mite, Demodex musculi, results in epithelial proliferation and aberrant hair follicle morphology accompanied by loss of stable commensalism with massive infestation and expansion of inflammatory ILC2s and other immune cells. Topical anti-parasitic agents, but not broad-spectrum antibiotics, reversed the phenotype. Commensal Demodex colonization of mammalian hair follicles is ubiquitous, including in humans, revealing an unanticipated role for ILC2s and type 2 immunity for skin homeostasis in the normal environment. One Sentence SummaryDynamic regulation of skin physiology by Type 2 innate immunity regulates healthy commensalism by parasitic Demodex mites.

immunology↗

bioRxiv2021

Novel EGFRvIII-CAR transgenic mice for rigorous preclinical studies in syngeneic mice

BackgroundRigorous preclinical studies of chimeric antigen receptor (CAR) immunotherapy will require large quantities of consistent and high-quality CAR-transduced T (CART)-cells that can be used in syngeneic mouse glioblastoma (GBM) models. To this end, we developed a novel transgenic (Tg) mouse strain with a fully murinized CAR targeting epidermal growth factor receptor variant III (EGFRvIII). MethodsWe first established the murinized version of EGFRvIII-CAR and validated its function using a retroviral vector (RV) in C57BL/6J mice bearing syngeneic SB28 GBM expressing EGFRvIII. Next, we created C57BL/6J-background Tg mice carrying the anti-EGFRvIII-CAR downstream of a Lox-Stop-Lox cassette in the Rosa26 locus. We bred these mice with CD4-Cre Tg mice to allow CAR expression on T-cells and evaluated the function of the CART-cells both in vitro and in vivo. In order to inhibit immunosuppressive myeloid cells within SB28 GBM, we also evaluated a combination approach of CART and an anti-EP4 compound (ONO-AE3-208). ResultsBoth RV- and Tg-CART-cells demonstrated specific cytotoxic activities against SB28-EGFRvIII cells. A single intravenous infusion of EGFRvIII-CART-cells prolonged the survival of glioma-bearing mice when preceded by a lymphodepletion regimen with recurrent tumors displaying profound EGFRvIII loss. The addition of ONO-AE3-208 resulted in long-term survival in a fraction of CART-treated mice and those survivors demonstrated delayed growth of subcutaneously re-challenged both EGFRvIII+ and parental EGFRvIII- SB28. ConclusionOur new syngeneic CAR Tg mouse model can serve as a useful tool to address clinically relevant questions and develop future immunotherapeutic strategies. Importance of studyThe majority of preclinical studies evaluating CART therapy for GBM have utilized xenografts implanted into immunocompromised mice. Because the successful development of these strategies will depend on the understanding of critical interactions between therapeutic cells and the endogenous immune environment, it is essential to develop a novel immunocompetent system which allows us to study these interactions in a robust and reproducible manner. To this end, we created a Tg mouse strain in which all T-cells express a murinized EGFRvIII-CAR. T-cells derived from these mice demonstrated consistent CAR expression and EGFRvIII-specific cytotoxicity while traditional transduction with a CAR vector showed batch-to-batch variability. The syngeneic system also gave us the opportunity to evaluate a combination regimen with blockade of myeloid-derived suppressor cells. The Tg-CART mice represent a novel system for robust, and reproducible preclinical investigations.

immunology↗