Lung-resident type 1 conventional dendritic cells (cDC1s) are critical for initiating immune responses against foreign respiratory viruses and endogenous assaults like cancer cells. Importantly, the cDCs of each tissue display unique phenotypes and functions dictated by the environment they populate. However, our understanding of the factors that regulate cDC1 development and function in the lung is minimal. The cytokines, FMS-like tyrosine kinase 3 ligand (FLT3L), and granulocyte-macrophage stimulating factor (GM-CSF) are essential for the development of tissue-resident cDC1, but in in vitro systems, these factors are insufficient to generate cDC1s with a lung-specific phenotype. RNAseq analysis of lung cDC1 identified a clear enrichment in genes associated with transforming growth factor beta (TGF-β) signaling, a factor also known to be enriched in the airspaces of the lung. TGF-β has been previously reported to maintain DC quiescence in the lung, however, our data suggested it may also play a role in differentiation and lung specification of these cells. Based on this, I hypothesized that adding TGF-β to in vitro bone marrow (BM) cultures alongside established cytokines FLT3L and GM-CSF would generate cDC1s phenotypically and functionally similar to in vivo lung cDC1s. The addition of TGF-β, but not GM-CSF, to optimized FLT3L-BMDC cultures resulted in development of cDC1 which highly resemble lung cDC1. The development of a high throughput in vitro system that differentiates functional lung cDC1s will not only inform the in vivo factors required for development of these cells but also provides a useful platform for interrogation of lung cDC1 function and a stepping-off point to begin models for human lung cDC1 production and study.

Pseudogenes were originally thought to be non-functional gene duplications that can ultimately be transcribed. Recent research has suggested that pseudogenes may regulate the immune system by interacting with retinoic acid-inducible gene-I-like receptors (RIG-I-like receptors, or RLRs); RIG-I and melanoma differentiation-associated protein 5 (MDA5), key innate immune sensors that recognize viral RNA. During viral infection, these self-RNAs can amplify the RLR signal leading to more efficient virus clearance. However, the extent of pseudogene regulation on RLRs is understudied. Therefore, I aim to investigate the potential of pseudogenes as RLR regulators and the mechanisms by which they exert their immunoregulatory effects. I hypothesize that RN7SL pseudogenes, which are non-functional copies of the RN7SL gene caused by duplication and mutations, amplify the immune response during viral infection. I aim to test this hypothesis by identifying host-derived RN7SL pseudogenes during infection, characterizing pseudogenes that bind to RLRs, and determining their effects on RLR signaling pathways and viral replication. As of today, we have performed preliminary investigations suggesting that during infection with SARS-CoV-2, pseudogenes of the RN7SLs (henceforth 7SLps) are induced compared to uninfected cells. In addition, we observed that a subset of these 7SLps interact with the RLR MDA5 at homeostasis and during West Nile virus infection. I expect to observe that 7SLps are involved in a feed-forward loop during infection and RLR activation, such as a decrease in interferon beta (IFN-β) production and lack of interaction with signal recognition proteins to show they act independently of the RLR pathway. Through this work, I aim to gain a deeper understanding of the function of pseudogenes in amplifying innate immune responses to protect against virus infection and the potential therapeutic applications of targeting pseudogenes in disease treatment.

The mammalian immune system can mount distinct responses depending on the type of pathogen. In the intestine, infections with large parasites trigger a “type 2” immune response. This culminates in fluid secretion into the lumen and increased smooth muscle contractility that pushes the pathogen, intestinal parasites, through the digestive tract and out of the body. Epithelial tuft cells detect worms through their brush-like microvilli projecting from the cells into the lumen and express specific receptors that detect parasites. Once a worm is detected, tuft cells produce IL-25. Binding of IL-25 to receptors on ILC2s causes intestinal ILC2s to proliferate and secrete key cytokines that bind to receptors on intestinal epithelial stem cells and cause a remodeling of the intestinal epithelium. While tuft cells have previously only been associated with parasitic infection, a significant expansion of tuft cells was also found in the distal small intestine of tumor-bearing APC-min mice. In colorectal tumors, IL-25 was found to promote intratumoral ILC2s and suppress anti-tumor immunity, which led to increased tumorigenesis. We used immunofluorescence microscopy to visualize and quantify Flare25, a reporter of Il25 transcription, in tumors and healthy tissue of APC-min mice. The role of IL25 is therefore important to understand, however antibodies for IL25 have failed to detect the protein, so finding another way to visualize it would help determine the role IL25 has in the “weep & sweep” and tumorigenesis pathways. Here we used CRISPR to insert DNA encoding a hemagglutinin (HA) tag at the N-terminal of the IL-25 protein. The presence of the DNA insertion was confirmed through sequencing and genotyping, however we could not detect HA by immunofluorescence microscopy. These studies expand our analysis of Il25 mRNA in the small intestine, but further work is needed to understand why we could not detect HA-tagged IL-25 protein. 

Melanoma is the leading, most deadly, cause of skin cancer. The survival rate of melanoma was observed from 2011-2017 to drop to 68% for patients with regional spread, and less than 30% for distant, metastatic tumours. Treatment is possible, and therapies such as immune checkpoint blockade (ICB) have been developed, increasing 5-year survival rates to over 50%. Current research has just begun to understand the importance of the tumour microenvironment (TME) for cancer survival. To address the hypothesis that specific gene expression within the TME influences the depth and quality of T-cell infiltration, and thus is a tumour-intrinsic property, I will be investigating infiltration across different subtypes of melanoma to assess whether T-cell exclusion can be predicted by transcriptional signatures. Tumours can be classified as immune-inflamed, (a result of CD8 T-cell infiltration), immune-excluded (CD8 T-cells localized around the border), or as an immune desert, entirely void of CD8 T-cells. A better understanding of these phenotypical and genotypical distinctions, propensities, and consequences is integral to healthcare. By implementing clinically relevant in vivo models, I am able to rigorously investigate TME effects on immunotherapies. The development of innovative humanized mouse models of melanoma, using genetically engineered ‘MISTRG’ recipient mice, has allowed us to mimic an entirely functional human immune system to respond to human melanoma tumours in vivo. Results from my lab suggest that the positioning of T cells in the tumour microenvironment is a tumour-intrinsic property, and modelling of the difference between infiltrated (hot) and excluded (cold) tumours is made possible with extensive modelling software, flow cytometry, epigenetics, and data analytics. The data that TME is intrinsic to patient success are thrilling, and future developments within the immunological field are sure to increase patient success even further. 

In humans, the KRAS protein normally acts as a switch to regulate cell growth. Acquisition of certain mutations result in constant activation of the protein, leading to uncontrollable cell growth. The most frequent such mutations are at the glycine-12 residue of the protein, including changes to valine (G12V). Cells with this mutation can be recognized and eliminated by T cells engineered to express an antigen-specific receptor (TCR), but posttranslational modifications, such as methylation, can interfere with the ability of such engineered T cells to recognize G12V mutant KRAS. The objective of my project is to identify and eventually disrupt mechanisms that lead to methylation of KRAS mutant antigens. I hypothesize specific enzymes are responsible for methylation of mutant KRAS. To accomplish this, I will generate CRISPR-Cas9 gene knockout libraries to screen for potentially responsible enzymes. The CRISPR library will target human genes encoding methyltransferase and demethylase enzymes. I will co-culture these CRISPR-Cas9 knockout cancer cells with T cells engineered with a TCR targeting the unmethylated KRAS mutant antigen. From the tumor cells that have survived this coculture, I will sequence genomic DNA to determine which knockouts are enriched/depleted after the co-culture. Preliminary results show certain gene knockouts are significantly enriched/depleted in co-culture compared to baseline. The sgRNAs that appear from the high-throughput knockout library most likely to be involved in the KRAS methylation pathway will be individually evaluated in additional co-culture experiments. This should allow me to assess the proliferation/susceptibility of these cancer cells in more detail. For confirmation of mechanism, I will evaluate the methylation status of the KRAS mutant antigen in cancer cells rendered susceptible. My goal is to determine the process leading to methylation of KRAS antigen and then target it to allow for more effective targeting of KRAS-driven cancers using TCR-T cell immunotherapy.