Inborn errors of immunity (IEI) are congenital immune system dysfunctions that are often caused by monogenic defects. The Butte Lab recently identified a patient with abnormally low T cell counts leading to severe immunodeficiencies. While defects in thymic development is a possible explanation, an alternative hypothesis is that the patient’s symptoms arise from a mutation in the VAV1 gene which was identified through whole genome sequencing. The patient carries a heterozygous Q697R variant of unknown significance. VAV1 encodes a guanine nucleotide exchange factor that plays a critical role in T cell receptor (TCR) signaling. The objective of this study is to assess the consequences of Vav1 loss by comparing calcium flux responses, protein phosphorylation, CD69 expression, and cytokine production between WT and experimental groups. Preliminary calcium flux measurements revealed that WT Jurkat cells displayed an approximately four-fold increase in intracellular calcium levels following anti-CD3 stimulation whereas J.Vav1 cells, Jurkat cells lacking Vav1, showed no detectable calcium response which indicates impaired TCR-mediated signaling. At the moment, we have successfully generated plasmids encoding both WT and Q697R Vav1 and electroporated them into J.Vav1 cells. Understanding the functional impact of this Vav1 variant may provide insight into mechanisms of immunodeficiency and inform potential therapeutic strategies to regulate T and B cell-mediated autoimmunity and chronic inflammation.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common chronic liver condition, affecting over 25% of adults globally. MASLD presents as a lipid accumulation in at least 5% of hepatocytes and at least one feature of insulin resistance. Men experience MASLD at higher prevalence, a more rapid progression and more severe pathology than women. Using a Four Core Genotypes (FCG) mouse model, the Reue Lab has found a gonadal sex difference in the hepatic lipid morphology in mice induced to develop MASLD: XX and XY mice with testes show hepatocytes with many small lipid droplets, while XX and XY mice with ovaries show hepatocytes with few large lipid droplets. We seek to understand whether the sex difference in lipid droplet formation influences the difference seen in MASLD. We identified a substantial sex difference in hepatic levels of Calsyntenin-3β (CLSTN3B), a protein involved in lipid droplet formation and morphology in adipocytes. In C57BL/6J male and female and FCG mice, liver CLSTN3B mRNA and protein levels are elevated in males and mice with testes. Using gonadectomized mice, we demonstrated that acute effects of testicular hormones likely regulate the sex difference in CLSTN3B levels and lipid droplet morphology. To test whether CLSTN3B directly influences lipid droplets, an overexpression of Clstn3b in hepatocytes is currently underway. An understanding of the Clstn3b pathway and its role in the sex difference of MASLD progression may help identify new therapeutic targets for MASLD.

Congenital limb defects are among the most common developmental abnormalities, yet early human limb development remains poorly understood because embryonic tissue is difficult to access. Human embryonic stem cell (hESC)-based models offer a powerful platform for studying these early events in vitro. Limb development begins when lateral plate mesoderm (LPM) gives rise to mesenchymal cells that form the limb bud and induce the overlying ectoderm to become the apical ectodermal ridge (AER), a key signaling center for limb outgrowth and patterning. In the Pyle Lab, a 3D hESC-derived limb bud organoid enriched for LPM-like cells has been established, but it currently lacks AER tissue. My project asks whether integrating LPM and AER tissues can generate a more representative 3D human limb bud model. Using H9 hESCs, I will apply defined small molecules and growth factors to induce coordinated LPM and AER differentiation in 3D organoids. I will assess lineage identity and tissue organization using RT-qPCR and immunofluorescence for developmental markers including HAND1 and TP63. I expect this work to produce an integrated human limb bud model with improved cellular complexity and developmental relevance. This project will advance a human-specific system for studying limb development, provide a novel model for testing therapies, and offer a foundation for investigating the origins of congenital limb disorders.

Mild hyperthermia is emerging as a promising strategy to enhance anti-tumor immunity, yet its effects on immune signaling within the hepatocellular carcinoma tumor microenvironment remain poorly defined. This project investigated how sustained mild hyperthermia modulates transcriptional changes associated with anti-tumor immune responses in hepatocellular carcinoma cells. To address this question, cultured HCC cell lines were exposed to controlled mild hyperthermic conditions, after which gene expression changes were assessed using bulk RNA sequencing data generated in the laboratory. Differential expression and pathway enrichment analyses revealed modulation of immune-related signaling networks, including interferon pathways, antigen presentation machinery, and stress response regulators. Notably, trends in immune checkpoint-associated gene expression suggested the presence of concurrent immunoregulatory responses. These findings provide insight into the molecular mechanisms through which thermal modulation influences tumor-immune interactions and highlight the potential of mild hyperthermia as an adjuvant strategy to improve immunotherapeutic outcomes in hepatocellular carcinoma.

Cystic Fibrosis (CF) is an autosomal disease affecting over 100,000 individuals worldwide and can result from more than 2,000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Current treatments include modulator therapies that partially restore CFTR protein function in epithelial cells of the airways, sweat glands, pancreas, gastrointestinal tract, and reproductive system. However, no approved therapies exist for Class I mutations, which produce no functional CFTR protein. We investigated restoring the wild-type sequence in the W1282X Class I mutation using adenine base editing. K562 cell lines harboring the mutation were generated via bulk transfection and fluorescence-activated cell sorting. Multiple adenine base editors (ABEMAX, ABE8.20, UMAX-rest1, UMAX-ex1, UMAX-ex2) and guide RNAs were tested. Editing efficiency was optimized through electroporation across varying plasmid doses, systematically evaluating base editor and guide RNA combinations. Across conditions from 1.33 µg to 6.66 µg total plasmid, 2.66 µg (0.66 µg guide RNA, 2.00 µg base editor) yielded the best results, achieving 44% on-target and 37% bystander editing. The g6-ABEMAX combination performed best, with 40% on-target and 22% bystander editing. UMAX editors underperformed relative to ABEMAX. These findings suggest W1282X can be corrected to wild-type or converted to a modulator-responsive Class II mutation, supporting a potential therapeutic strategy for Class I CF mutations.