Soil-transmitted helminths impact over 1.5 billion people worldwide, disproportionately affecting school-aged children and pregnant women. Hosts issue a “weep and sweep” Type II immune response to expel helminth parasites from the intestines. Ruptured epithelial cells secrete the cytokine interleukin (IL)-33, which recruits innate lymphoid type 2 cells (ILCs)2 and CD4+ T-helper type 2 (Th2) cells. ILC2s release IL-13, encouraging stem cell differentiation into tuft and goblet cells to facilitate tissue repair and worm expulsion. Anemia is prevalent in helminth-rich environments because of elevated rates of malaria and malnutrition. Despite correlations between anemia and helminth infection, the impact of anemia on the Type II immune response in the small intestines remains unknown. Using N. brasiliensis (Nb), a bloodsucking hookworm-like parasite, I observed that one week post infection, iron-deficient (ID) mice were less capable of expelling worms compared to iron-sufficient (IS) mice. Through tuft cell immunofluorescence staining in the small intestine, I observed comparable hyperplasia in IS and ID-Nb infected mice but noticed differences in cell localization: ID-Nb infected mice had decreased numbers of tuft cells in the crypts compared to IS-Nb mice. This suggests that ID Nb-infected mice could be experiencing decreased migration/proliferation of tuft cells, compared to their IS-infected counterparts. Using EdU, a synthetic nucleotide tag that labels newly synthesized DNA, we can understand cellular proliferation patterns in IS vs. ID Nb mice. Co-staining for tuft cells permits us to merge events and track unique vs. universal trends in cell behavior, including cells’ migration patterns. I hypothesize that ID-Nb mice will have decreased cellular proliferation and migration compared to IS-Nb mice, ultimately impacting worm burden. These findings offer insights into the mechanism behind negative outcomes in anemic hosts, and could contribute to dietary intervention or therapies targeting the epithelium to alleviate burdens of helminth infection.

Acetylcholine (ACh), a neurotransmitter known for its roles in neuromuscular function and cognition, has recently been implicated in immune regulation, particularly in the context of Type-2 immunity. The Type-2 response combats parasites at mucosal and cutaneous sites and plays a role in allergic diseases like asthma and food allergy. In the intestine, Type-2 inflammation involves a dramatic remodeling of the intestinal epithelium via the activation of intestinal epithelial stem cells (ISCs), which results in the hyperplasia of specialized effector-like secretory cells such as goblet and tuft cells. These epithelial cells then produce factors that talk back to the epithelium, such as Ach, and factors that promote Type 2 immune responses, such as the cytokine interleukin-25. Tuft cells are the only intestinal epithelial cells that express choline acetyltransferase, the enzyme for ACh synthesis. Recent work suggests that during helminth infection, tuft cells release ACh in response to IL-13 signaling, implying a role for ACh in regulating epithelial responses. However, its specific function in epithelial remodeling and Type-2 immunity remains unclear.This project aims to investigate ACh’s epithelial intrinsic role in Type-2 immune responses using an in-vitro enteroid model of the intestinal epithelium. Enteroids, 3-D cultures derived from stem cells, model the epithelium without immune cells, allowing for a focused examination of epithelial-intrinsic factors in immune responses. By culturing and treating enteroids from wild-type and tuft cell-deficient mice with ACh, we will assess its effects on goblet cell proliferation, inflammation, and stem cell renewal during injury regeneration responses. I hypothesize that ACh enhances the pro-Type-2 inflammatory response in enteroid epithelial cells, leading to heightened immune activity. This research will advance our understanding of the neuro-immune axis in the gut and may have implications for parasitic infections and allergic inflammation.

Regulatory T cells (Tregs) play an integral role in self-tolerance and the preventing autoimmunity by suppressing the immune response. As such, inducing Treg expansion is a promising avenue for treating autoimmunity. Previous studies have shown that treatment with an interleukin-2 (IL-2) mutein Fc.Mut24 causes more robust Treg expansion than wildtype IL-2 and is effective at preventing autoimmunity in the non-obese diabetic (NOD) mouse model. In this study, we identified the synergistic roles of the T cell receptor (TCR) and IL-2 receptor in IL-2 Fc.Mut24-mediated Treg expansion, leading to the question of how TCR stimulation is affected during treatment. Classical dendritic cells (cDCs) present antigens to the TCR on Tregs, leading to an increased Treg population to suppress autoimmunity. This study aims to elucidate the role of interactions between Tregs and cDCs during IL-2 Fc.Mut24 treatment using in vivo mouse models. Through the uLIPSTIC (universal labelling immune partnerships by sortagging intracellular contacts) model, we reveal the effect of IL-2 Fc.Mut24 on the frequency of short-range and transient Treg/cDC interactions. Using Zbtb46-DtR bone marrow chimeras to deplete cDCs, we may begin to understand the extent to which cDCs are necessary for the robust Treg expansion typically seen during IL-2 Fc.Mut24 treatment. Preliminary results of cDC depletion do show reduced Treg expansion. We expect to find that IL-2 Fc.Mut24 promotes immunosuppressive Treg/cDC interactions in vivo, as indicated by increased frequency of interaction and cDC-dependent Treg expansion. The findings from this study will contribute to a greater understanding of how IL-2 mutein therapies mechanistically combat autoimmunity, potentially paving the way for identifying new applications to treat disease.

Adoptive cell therapy with CAR-T cells has shown promise in hematological malignancies, but efficacy in solid tumors remains a challenge in part due to CAR-T cell exhaustion and antigen heterogeneity. However, the vast majority of preclinical models do not recapitulate the tumor-immune interactions that produce these barriers. To study CAR-T therapy in a rigorous model that recapitulates tumor-immune barriers, we adapted a Kras^LSL-G12D/+;P53^f/f (KP) genetically engineered mouse model (GEMM) of lung adenocarcinoma. However, adapting the KP-GEMM model for various target antigens, genetic drivers of disease, or interfacing with the vast array of powerful genetic mouse models is resource intensive which prohibits widespread utility. Here, we propose a defined, modular system for generating GEMM for CAR-T preclinical studies using the Sleeping Beauty (SB) transposon system. The proposed system uses polyethylenimine (PEI) to deliver SB transposon encoding oncogenic Kras^G12D and P53^R175H dominant alleles as well as our target antigen hROR1, in vivo to wild-type mice. We demonstrate that in vitro PEI successfully introduces genetic cargo into lung epithelial cell lines, while SB transposons mediate stable integration and expression. Next, we will test this in vivo. This system affords the induction of tumors with specific oncogenic driver mutations and specific tumor antigens on any genetic background. Ultimately, we expect that this approach will streamline preclinical use of GEMM in preclinical research. 

In Adoptive Cell Therapy (ACT), a novel modality of cancer therapy, immune cells can be engineered with T cell receptors (TCRs) to aid in targeting specific antigens presented on the surface of cancer cells. However, TCR-T cells often have limited persistence after transfer into patients, which has hampered the effectiveness of this therapy for solid tumors. Last year, our lab identified LSD1 as a target for drug inhibition, which is an enzyme that alters the epigenome of cells via histone modifications. My project aims to understand the mechanism of LSD1 inhibitor drugs, as well as the effect of these drugs on two types of T cells: cytotoxic CD8+ cells and helper CD4+ cells. In addition to understanding how LSD1 drugs work, I also ask exactly how CD4+ cells enhance the function of CD8+ cells in tumor killing. Which receptors on CD8+ cells are activated by helper T cells, what is the signal phosphorylation pathway transducing the "helping" signal from receptors, and what downstream epigenetic regulators play a role in translating the "helping" signal into better function in CD8+ T cells? To assess these interactions, I will generate a diverse population of CD8+ T cells with targeted receptor knockouts, known as a receptor library. Similar libraries will be generated for epigenetic regulators as well as kinases/phosphatases. The performance of T cells will be assessed via coculture assays, where T cells can kill tumor cells but not fully eliminate the tumor because of periodic addition of new tumor cells. At the end of the coculture period, we will assess gRNAs enriched in dysfunctional populations, which will identify genes critical to CD8+ T cell function. This project aims to provide enhanced function of T cells that are better suited for applications in clinic.

Type 2 immunity is the immune response activated by allergens and parasites, and recently type 2 immune cells were discovered to have pro-tumor functions. We are working to understand how tumors activate and regulate a type 2 immune response using a mouse model (Apc^Min/+) which mimics pre-malignant human colorectal cancer. This project focuses specifically on the role of mast cells; a key component of type 2 immunity attributed with both pro- and anti-tumor properties. Mast cells are known to infiltrate tumors, and previous work in the lab has shown that the intestinal epithelial cytokine IL-33 promotes activation of Apc^Min/+ tumor-associated mast cells leading to a pro-tumor response. We hypothesized that mast cells and IL-33 would be colocalized in these tumors because of this association. While immunofluorescence (IF) imaging and reverse transcriptase, quantitative polymerase chain reaction (RT-qPCR) results confirmed an upregulation of IL-33 in tumors, IF data did not support the colocalization hypothesis. To further understand the role mast cells may have in type 2 immune response activation, the antibody c-Kit was used to deplete mast cells from Apc^Min/+ mice and I compared expression of the enzyme Arginase 1 and cytokine IL-13 to control Apc^Min/+ mice using qPCR. These targets both serve as measurements of type 2 immune response “outputs”. Data showed a decrease for both targets in the mast cell-depleted mice, which supports the idea that mast cells have a role in activating the type 2 immune response in tumors. Additionally, we made a novel observation of intraepithelial mast cells residing within the tightly packed epithelial tumor cells, which is one way we have come to better understand the morphology of the Apc^Min/+ tumors. We continue to explore where cells and molecules are located in these tumors because this facilitates our thinking about how interactions may be taking place.

Lentivirus is a well-established gene-editing tool commonly used in cellular research. Recently, its widespread adoption has led to the development of numerous protocols for the transduction of primary human T-cells. However, generating high-titer virus for large vectors remains a challenge, and there is a need for optimized protocols – particularly for creating Base Editor lentivirus for a 15 kb vector. By developing a method to estimate transduction efficiency in primary human T-cells using viral titers, significant reduction to the waste of valuable human samples could be achieved. To address these challenges, we tested various variables—including plasmid concentration, media formulations, and transfection reagents—within infection protocols to optimize lentivirus production and improve T-cell transduction efficiency. By refining the protocol for creating Base Editor lentivirus, we aim to base-edit autoimmune-associated variants in human CD4 T-cells and assess their impact on T-cell effector function. This work is crucial for advancing base editing technologies in the Ray Lab and will contribute to the broader field of immunology.