Faculty Bibliography

Loss-of-function mutations in KEAP1 frequently occur in lung cancer and are associated with poor prognosis and resistance to standard of care treatment, highlighting the need for the development of targeted therapies. We previously showed that KEAP1 mutant tumors consume glutamine to support the metabolic rewiring associated with NRF2-dependent antioxidant production. Here, using preclinical patient-derived xenograft models and antigenic orthotopic lung cancer models, we show that the glutamine antagonist prodrug DRP-104 impairs the growth of KEAP1 mutant tumors. We find that DRP-104 suppresses KEAP1 mutant tumors by inhibiting glutamine-dependent nucleotide synthesis and promoting antitumor T cell responses. Using multimodal single-cell sequencing and ex vivo functional assays, we demonstrate that DRP-104 reverses T cell exhaustion, decreases T_regs, and enhances the function of CD4 and CD8 T cells, culminating in an improved response to anti-PD1 therapy. Our preclinical findings provide compelling evidence that DRP-104, currently in clinical trials, offers a promising therapeutic approach for treating patients with KEAP1 mutant lung cancer.

Distinct subsets of T lymphocytes express CX3CR1 under inflammatory conditions, but little is known about CX3CR1+CD4+ T cells during type 2 inflammation in helminth infections. In this study, we used a fate-mapping mouse model to characterize CX3CR1+CD4+ T cells during both acute Nippostrongylus brasiliensis and chronic Schistosoma mansoni murine models of helminth infections, revealing CX3CR1+CD4+ T cells to be an activated tissue-homing subset with varying capacity for cytokine production. Tracking these cells over time revealed that maintenance of CX3CR1 itself along with a TH2 phenotype conferred a survival advantage in the inflamed tissue. Single-cell RNA sequencing analysis of fate-mapped CX3CR1+CD4+ T cells from both the peripheral tissue and the spleen revealed a considerable level of diversity and identified a distinct population of BCL6+TCF-1+PD1+CD4+ T cells in the spleen during helminth infections. Conditional deletion of BCL6 in CX3CR1+ cells resulted in fewer CX3CR1+CD4+ T cells during infection, indicating a role in sustaining CD4+ T cell responses to helminth infections. Overall, our studies revealed the behavior and heterogeneity of CX3CR1+CD4+ T cells during type 2 inflammation in helminth infections and identified BCL6 to be important in their maintenance.

Lung cancer treatment has benefited greatly through advancements in immunotherapies. However, immunotherapy often fails in patients with specific mutations like KEAP1, which are frequently found in lung adenocarcinoma. We established an antigenic lung cancer model and used it to explore how Keap1 mutations remodel the tumor immune microenvironment. Using single-cell technology and depletion studies, we demonstrate that Keap1-mutant tumors diminish dendritic cell and T cell responses driving immunotherapy resistance. This observation was corroborated in patient samples. CRISPR-Cas9-mediated gene targeting revealed that hyperactivation of the NRF2 antioxidant pathway is responsible for diminished immune responses in Keap1-mutant tumors. Importantly, we demonstrate that combining glutaminase inhibition with immune checkpoint blockade can reverse immunosuppression, making Keap1-mutant tumors susceptible to immunotherapy. Our study provides new insight into the role of KEAP1 mutations in immune evasion, paving the way for novel immune-based therapeutic strategies for KEAP1-mutant cancers.

Genetically engineered mouse models (GEMMs) help us to understand human pathologies and develop new therapies, yet faithfully recapitulating human diseases in mice is challenging. Advances in genomics have highlighted the importance of non-coding regulatory genome sequences, which control spatiotemporal gene expression patterns and splicing in many human diseases^1,2. Including regulatory extensive genomic regions, which requires large-scale genome engineering, should enhance the quality of disease modelling. Existing methods set limits on the size and efficiency of DNA delivery, hampering the routine creation of highly informative models that we call genomically rewritten and tailored GEMMs (GREAT-GEMMs). Here we describe 'mammalian switching antibiotic resistance markers progressively for integration' (mSwAP-In), a method for efficient genome rewriting in mouse embryonic stem cells. We demonstrate the use of mSwAP-In for iterative genome rewriting of up to 115 kb of a tailored Trp53 locus, as well as for humanization of mice using 116 kb and 180 kb human ACE2 loci. The ACE2 model recapitulated human ACE2 expression patterns and splicing, and notably, presented milder symptoms when challenged with SARS-CoV-2 compared with the existing K18-hACE2 model, thus representing a more human-like model of infection. Finally, we demonstrated serial genome writing by humanizing mouse Tmprss2 biallelically in the ACE2 GREAT-GEMM, highlighting the versatility of mSwAP-In in genome writing.

Our previous studies identified a population of stem cell-like proliferating myeloid cells within inflamed tissues that could serve as a reservoir for tissue macrophages to adopt different activation states depending on the microenvironment. By lineage-tracing cells derived from CX3CR1+ precursors in mice during infection and profiling by single-cell RNA sequencing, in this study, we identify a cluster of BIRC5+ myeloid cells that expanded in the liver during chronic infection with either the parasite Schistosoma mansoni or the bacterial pathogen Staphylococcus aureus. In the absence of tissue-damaging toxins, S. aureus infection does not elicit these BIRC5+ cells. Moreover, deletion of BIRC5 from CX3CR1-expressing cells results in improved survival during S. aureus infection. Hence the combination of single-cell RNA sequencing and genetic fate-mapping CX3CR1+ cells revealed a toxin-dependent pathogenic role for BIRC5 in myeloid cells during S. aureus infection.

Chikungunya virus (CHIKV) infection has been associated with severe cardiac manifestations, yet, how CHIKV infection leads to heart disease remains unknown. Here, we leveraged both mouse models and human primary cardiac cells to define the mechanisms of CHIKV heart infection. Using an immunocompetent mouse model of CHIKV infection as well as human primary cardiac cells, we demonstrate that CHIKV directly infects and actively replicates in cardiac fibroblasts. In immunocompetent mice, CHIKV is cleared from cardiac tissue without significant damage through the induction of a local type I interferon response from both infected and non-infected cardiac cells. Using mice deficient in major innate immunity signaling components, we found that signaling through the mitochondrial antiviral-signaling protein (MAVS) is required for viral clearance from the heart. In the absence of MAVS signaling, persistent infection leads to focal myocarditis and vasculitis of the large vessels attached to the base of the heart. Large vessel vasculitis was observed for up to 60 days post infection, suggesting CHIKV can lead to vascular inflammation and potential long-lasting cardiovascular complications. This study provides a model of CHIKV cardiac infection and mechanistic insight into CHIKV-induced heart disease, underscoring the importance of monitoring cardiac function in patients with CHIKV infections.

In early life, susceptibility to invasive infection skews toward a small subset of microbes, whereas other pathogens associated with diseases later in life, including Streptococcus pneumoniae (Spn), are uncommon among neonates. To delineate mechanisms behind age-dependent susceptibility, we compared age-specific mouse models of invasive Spn infection. We show enhanced CD11b-dependent opsonophagocytosis by neonatal neutrophils improved protection against Spn during early life. The augmented function of neonatal neutrophils was mediated by higher CD11b surface expression at the population level due to dampened efferocytosis, which also resulted in more CD11b^hi "aged" neutrophils in peripheral blood. Dampened efferocytosis during early life could be attributed to the lack of CD169⁺ macrophages in neonates and reduced systemic expressions of multiple efferocytic mediators, including MerTK. On experimentally impairing efferocytosis later in life, CD11b^hi neutrophils increased and protection against Spn improved. Our findings reveal how age-dependent differences in efferocytosis determine infection outcome through the modulation of CD11b-driven opsonophagocytosis and immunity.

Small animal models have been a challenge for the study of SARS-CoV-2 transmission, with most investigators using golden hamsters or ferrets. Mice have the advantages of low cost, wide availability, less regulatory and husbandry challenges, and the existence of a versatile reagent and genetic toolbox. However, adult mice do not robustly transmit SARS-CoV-2. Here we establish a model based on neonatal mice that allows for transmission of clinical SARS-CoV-2 isolates. We characterize tropism, respiratory tract replication and transmission of ancestral WA-1 compared to variants Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Omicron BA.1 and Omicron BQ.1.1. We identify inter-variant differences in timing and magnitude of infectious particle shedding from index mice, both of which shape transmission to contact mice. Furthermore, we characterize two recombinant SARS-CoV-2 lacking either the ORF6 or ORF8 host antagonists. The removal of ORF8 shifts viral replication towards the lower respiratory tract, resulting in significantly delayed and reduced transmission in our model. Our results demonstrate the potential of our neonatal mouse model to characterize viral and host determinants of SARS-CoV-2 transmission, while revealing a role for an accessory protein in this context.

Macrophages facilitate critical functions in regulating pathogen clearance and immune homeostasis in tissues. The remarkable functional diversity exhibited by macrophage subsets is dependent on tissue environment and the nature of the pathological insult. Our current knowledge of the mechanisms that regulate the multifaceted counter-inflammatory responses mediated by macrophages remains incomplete. Here, we report that CD169+ macrophage subsets are necessary for protection under excessive inflammatory conditions. We show that in the absence of these macrophages, even under mild septic conditions, mice fail to survive and exhibit increased production of inflammatory cytokines. Mechanistically, CD169+ macrophages control inflammatory responses via interleukin-10 (IL-10), as CD169+ macrophage-specific deletion of IL-10 was lethal during septic conditions, and recombinant IL-10 treatment reduced lipopolysaccharide (LPS)-induced lethality in mice lacking CD169+ macrophages. Collectively, our findings show a pivotal homeostatic role for CD169+ macrophages and suggest they may serve as an important target for therapy under damaging inflammatory conditions.

Small animal models have been a challenge for the study of SARS-CoV-2 transmission, with most investigators using golden hamsters or ferrets ^1,2 . Mice have the advantages of low cost, wide availability, less regulatory and husbandry challenges, and the existence of a versatile reagent and genetic toolbox. However, adult mice do not transmit SARS-CoV-2 ³ . Here we establish a model based on neonatal mice that allows for transmission of clinical SARS-CoV-2 isolates. We characterize tropism, respiratory tract replication and transmission of ancestral WA-1 compared to variants alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2) and omicron (B.1.1.529). We identify inter-variant differences in timing and magnitude of infectious particle shedding from index mice, both of which shape transmission to contact mice. Furthermore, we characterize two recombinant SARS-CoV-2 lacking either the ORF6 or ORF8 host antagonists. The removal of ORF8 shifts viral replication towards the lower respiratory tract, resulting in significantly delayed and reduced transmission. Our results demonstrate the potential of our neonatal mouse model to characterize viral and host determinants of SARS-CoV-2 transmission, while revealing for the first time a role for an accessory protein this context.