HMS Theses and Dissertations

This master’s thesis on improving genetic diagnosis revolves around two projects. Project 1: Participant Perspectives on Rare Disease Genomic Research Abstract Introduction: Although advances in genomic sequencing technology have improved diagnostic capabilities, access to these technologies is variable. Furthermore, novel approaches to genomic analysis may be outside of the scope of usual clinical testing. Rare disease genomic research thus represents a potential pathway to genetic diagnosis. However, factors influencing the decision to participate in a genomic research study remain incompletely understood. We therefore investigated motivators for participation in a rare disease genomic research study and the impact of diagnostic findings. Methods: Prospective quantitative analysis of survey data from participants in the Rare Genomes Project was conducted. Surveys were sent upon enrollment to the study and at 3 and 12 months after result disclosure and included both novel items and the Perceived Stress Scale. Results: 509 participants responded out of 884 targeted surveys, either affected persons (226) or their caregivers (283). For all respondents, diagnostic importance, improved clinical understanding, future preparedness, and treatment optimization were the strongest motivators for seeking a genetic diagnosis. Perceived stress upon entry to the study was significantly higher than population norms (17.61 vs 13, P 0.00001). Variables associated with perceived diagnostic importance are mainly related to the financial burden of health conditions (OR 3.859 (95% CI 1.306–11.404), P = 0.015). At follow-up timepoints, diagnosed participants endorsed statements related to a positive impact of the diagnosis on group connections. Perceived benefit from social connectedness became more prominent over time. Conclusion: We described the psychosocial context of research enrollment for a diverse cohort of rare disease genomic research participants and identified financial problems as the prominent determinant for diagnostic importance perception. We also highlighted a potential positive impact of the genetic diagnosis in reducing the perceived stress and shifting feelings towards genetic diagnosis from vigilance to acceptance.

Project 2: Analysis of Splicing Coding and Noncoding Genes and Pseudogenes Reveals Novel Gene-Disease Relationships Abstract Splicing is a complex molecular mechanism occurring in each living human cell where the precursor messenger RNA (pre-mRNA) is processed into the mature messenger RNA (mRNA). It involves more than 300 protein-coding genes (PCGs) and around 43 small nuclear RNA (snRNA) genes. However, fewer than 30 diseases have been described as spliceosomopathies to date. This discrepancy points towards splicing machinery as an underexplored area for genetic discoveries. Furthermore, there are almost 1700 spliceosome-related snRNAs pseudogenes that are overlooked in diagnostic analyses, but recently a novel gene-disease relationship has led to reclassification of a snRNA pseudogene to a gene associated with a developmental and epileptic encephalopathy, highlighting the importance of further evaluation of snRNA pseudogenes. We evaluated both coding and noncoding snRNA genes and their pseudogenes using adapted approaches to each gene category. Analysis strategies involved evaluating heterozygous variants in PCGs with loss of function constraint (pLI > 0.9) and in snRNAs in variant depleted regions in gnomAD, with biallelic variants reviewed throughout both gene sets. For snRNA pseudogenes, we prioritized candidates having similar epigenomic, genomic, and hypermutability features to functional snRNA genes. These signals, located upstream, downstream, and within a gene, indicate the likelihood of it being a functional gene. For around 23,000 families with rare disease sequenced through the GREGoR consortium, we identified 30 variants of interest across 9 PCGs with established gene-disease relationships (GDRs) and 13 genes not yet associated with disease, including one pseudogene. Of note, one gene, CWC25, had candidate variants with dominant and recessive inheritance patterns. For snRNAs genes, we identified 55 variants of interest located in seven established GDR and 11 genes not yet associated with disease, including two pseudogenes, prioritized by one or two of the prioritization strategies. Our results expanded the phenotypic expression of one variant located in the three-way junction in RNU4-2 gene from retinal dystrophy (RD) to RD and Neurodevelopmental disorder (NDD). We also suggested pleiotropy expansion for RNU6 variants with NDD and oculo-muscular fibrosis. Finally, we identified biallelic variants in RNU5 family, suggesting inheritance pattern extension. This study highlighted the importance of splicing-related PCG and snRNA in rare disease diagnosis. The genes prioritized through this research represent selected candidates for future confirmatory studies.

Chimeric antigen receptor (CAR) T-cell therapy has transformed the treatment of hematologic malignancies, but its application remains limited by manufacturing constraints, toxicity, and antigen escape. CAR-engineered natural killer (CAR-NK) cells represent a promising alternative due to their favorable safety profile, reduced risk of graft-versus-host disease, and capability for off-the-shelf use. However, a main limitation of CAR-NK therapy is poor in vivo persistence, largely due to dependence on exogenous cytokine support. This study addresses this challenge by developing a targeted cytokine delivery strategy, the CAR-Enhancer (CAR-E) platform on NK cells. CAR-Es are bifunctional molecules composed of a CAR-targeting antigen domain fused to a low-affinity mutant cytokine, enabling selective delivery of cytokine signaling exclusively to CAR-expressing NK cells. This design concentrates survival and activation signals at the CAR interface while minimizing systemic toxicity and off-target immune activation. First, we demonstrate that IL-2–based CAR-E constructs selectively bind CAR-NK cells and induce potent STAT5 signaling in a CAR-dependent manner, while sparing non-transduced immune cells. This targeted signaling results in preferential expansion and functional activation of CAR-NK cells. Functionally, CAR-E enhances CAR-NK cytotoxicity against tumor targets without impairing CAR-antigen engagement. Notably, CAR-E also boosts NK-intrinsic cytotoxic pathways, enabling enhanced tumor killing even in the absence of canonical CAR signaling. This dual mechanism demonstrates a broader immunomodulatory role for CAR-E, beyond traditional CAR-mediated targeting. In vivo, exogenous CAR-E administration promotes robust CAR-NK cell expansion, multi-organ persistence, and complete tumor control in a xenograft model of multiple myeloma. Persisting CAR-NK cells retain an activated, non-exhausted phenotype and preserved cytotoxic function, demonstrating durable functional competence. To further improve translational feasibility, we engineered armored CAR-NK cells that autonomously secrete CAR-E via a bicistronic T2A construct. This self-secretion system increases cytotoxicity, metabolic fitness, and resistance to exhaustion under chronic tumor exposure. In vivo, armored CAR-NK cells demonstrate prolonged persistence and improved tumor control even when tumor challenge is delayed, showing their potential for long-term immunosurveillance. This work confirms CAR-E as a novel and versatile strategy to enhance CAR-NK cell persistence and functionality. By enabling targeted cytokine delivery without systemic toxicity, the CAR-E platform addresses a central limitation of CAR-NK therapy and provides a broadly applicable framework for improving adoptive cell therapies in cancer immunotherapy.

NOD2 is a cytosolic pattern-recognition receptor that detects bacterial muramyl dipeptide (MDP) and activates inflammatory signaling through recruitment of receptor-interacting serine/threonine-protein kinase 2 (RIPK2). Genetic variants in NOD2 are strongly associated with Crohn’s disease and Blau syndrome, yet the molecular basis of receptor activation and adaptor engagement remains incompletely defined. In particular, the architecture of activated NOD2 and its coupling to RIPK2 assembly have not been structurally resolved. Here, I establish a recombinant platform for biochemical and structural interrogation of human NOD2 and RIPK2. Full-length and domain-truncated NOD2 constructs were expressed in insect cells and purified for downstream analysis. Biochemical and imaging experiments showed that NOD2 undergoes temperature- and nucleotide-dependent oligomerization in vitro. Negative-stain electron microscopy revealed formation of discrete rounded oligomeric particles following ATPγS incubation, whereas size-exclusion chromatography and mass photometry indicated that both full-length and ΔCARD NOD2 were predominantly monomeric under resting conditions, with minor higher-order populations. The ΔCARD construct exhibited improved biochemical homogeneity and yielded a high-resolution cryo-electron microscopy reconstruction of the NOD2 monomeric core. This structure showed strong agreement with computational predictions for the NACHT and LRR regions and provides a framework for future analysis of disease-associated mutations and ligand-responsive conformational change. In contrast, oligomeric assemblies of both full-length and ΔCARD NOD2 remained structurally heterogeneous, limiting high-resolution reconstruction of the activated state. RIPK2 was also reconstituted in vitro and shown to form filamentous assemblies after tag removal. Biochemical assays, including crosslinking and native gel analysis, supported physical association between NOD2 and RIPK2, although a stable and structurally defined signaling supercomplex was not resolved. In THP-1 macrophages, MDP stimulation promoted redistribution of NOD2 toward a membrane-associated fraction that remained recoverable after detergent extraction, consistent with activation-dependent spatial reorganization. Together, these findings establish a tractable biochemical and structural framework for studying NOD2 activation. The data support a model in which NOD2 contains an intrinsic assembly program within its NACHT-LRR core, while RIPK2 engagement and higher-order signaling complex formation remain condition-dependent and structurally unresolved. This work provides a foundation for future studies of ligand binding, adaptor recruitment, disease-associated mutation mechanisms, and therapeutic modulation of the NOD2 pathway.

Background and Objectives: Mast cells exist as two functionally distinct subtypes, MCT and MCTC, whose identity is shaped by microenvironmental signals during tissue maturation. TGF-β has been identified as an instructive signal for MCT differentiation, and prior work has demonstrated that MCTC can acquire aspects of MCT identity upon TGF-β exposure. Whether mature MCT identity is actively maintained by ongoing TGF-β signaling, the transcriptional and functional extent of plasticity in both subtypes, and the contribution of SMAD3 to these responses remain uncharacterized. Methods: MCT and MCTC were differentiated from peripheral blood CD34+ progenitors in vitro. To examine plasticity in mature cells, TGF-β was withdrawn from MCT and added to MCTC for one week prior to analysis. To interrogate canonical signaling, the SMAD3-specific inhibitor SIS3 was applied concurrently with TGF-β. Transcriptional remodeling was assessed by bulk RNA sequencing, functional plasticity by IgE-mediated cysteinyl leukotriene and prostaglandin D2 measurement, and surface phenotypic remodeling by flow cytometric profiling. Results: TGF-β withdrawal from MCT and addition to MCTC produced asymmetric transcriptional responses, with 713 and 1,799 differentially expressed genes, respectively, spanning granule component, eicosanoid biosynthesis, and receptor gene programs. TGF-β bidirectionally regulated IgE-mediated eicosanoid production across both subtypes. Certain features of MCT identity, including low MRGPRX2 surface expression, were resistant to TGF-β withdrawal, whilst others, including CD33 and eicosanoid biosynthetic capacity, required continuous TGF-β signaling for their maintenance. In MCTC, TGF-β addition induced broad transcriptional and functional remodeling alongside suppression of MCTC-enriched surface markers. SMAD3 inhibition selectively attenuated TGF-β-driven cysteinyl leukotriene production in MCT and integrin α2B expression in both subtypes, demonstrating context-dependent and mediator-specific SMAD3 contributions. Conclusion: Human mast cell subtype identity comprises both developmentally consolidated programs that are resistant to acute microenvironmental change and actively maintained programs requiring continuous TGF-β signaling. MCT and MCTC differ fundamentally in the architecture of this relationship, with MCTC exhibiting broader susceptibility to TGF-β-driven remodeling. These findings reframe mast cell subtype identity as a dynamically maintained state shaped by microenvironmental signals.

Obesity and type 2 diabetes (T2D) are major metabolic diseases characterized by chronic inflammation and dysregulated adipose tissue homeostasis. Visceral adipose tissue (VAT) serves as an immunological organ that integrates metabolic and inflammatory signals through dynamic interactions between immune and stromal cells. Regulatory T cells (Tregs) are enriched in lean VAT and contribute to metabolic homeostasis by suppressing inflammation and regulating the IL-33-expressing VAT mesenchymal stromal cells (VmSCs). Emerging evidence suggests that stromal cells establish specialized immunoregulatory niches that sustain Treg populations and glucagon-like peptide-1 receptor agonists (GLP-1RAs) are widely used for T2D with improved metabolic outcomes, yet the mechanisms governing stromal–immune crosstalk and GLP-1RA effects on VAT microenvironment remain poorly understood. In this study, we investigated how semaglutide alters the immune and stromal compartments within VAT and examined the role of the IL-33–VmSC–Treg axis in mediating these effects. We found that semaglutide increases IL-33+ VmSCs proliferation and modulation of VAT ST2+ Tregs at cellular levels. In addition, semaglutide shifted adipogenic stromal populations by reducing VmSC5 and restoring VmSC4 in vivo, with stronger effects under long-term high-fat diet. Despite these changes, direct effects of semaglutide on Tregs were minimal in vitro, suggesting that GLP-1RA–mediated modulation of VAT immunity occurs indirectly through the stromal niche. Together, these findings support a model in which stromal cells and Tregs form a dynamic regulatory circuit that is reshaped by metabolic interventions. Together, this work highlights the importance of the immune-stromal axis in VAT regulation, provides new insight into mechanisms linking metabolism and immune regulation in VAT, and suggests potential novel therapeutic strategies for metabolic diseases.

The importance of extrafollicular adaptive immune responses is evident across diverse disease states. However, the precise identity of the T and B lymphocyte subsets that drive dysregulation and pathogenic extrafollicular autoantibody production remains unclear in conditions such as lupus, chronic graft-versus-host disease, and IgG4-related disease. Prior studies in COVID-19 and HIV have shown that class-switched antibodies can arise in the absence of germinal center activity, supporting the idea that class switch recombination may occur outside the germinal center and could depend on alternative forms of CD4 T cell help. This project investigated CD4 T cells interacting with B cells outside the conventional germinal center response. T-B conjugates increased substantially following sheep red blood cell immunization, indicating that these interactions expand during an active immune response. Importantly, the B cells bound within these conjugates were IgD+, indicating that the interacting B cell population was unswitched and consistent with an early stage of the humoral response. In LCMV infection, analysis of interacting CD4 T cells showed a heterogeneous population that included T-bet+ and Foxp3+ cells, with relatively few Bcl6+ cells, suggesting that B cell-interacting CD4 T cells outside the germinal center are more diverse than previously appreciated. Further phenotypic analysis identified distinct ICOS+PSGL1- and ICOS+PSGL1+ CD4 T cell populations also present in the sorted T-B pairs. The ICOS+PSGL1- population aligned more closely with follicular helper features, whereas the ICOS+PSGL1+ population represented a broader Th1-focused helper population. Bulk RNA sequencing supported this distinction, showing that ICOS+PSGL1+ cells exhibit a mixed transcriptional program rather than a purely follicular helper identity. Together, these findings support the existence of a heterogeneous CD4 T cell population that engages unswitched B cells outside the classical germinal center pathway and may contribute to early extrafollicular B cell help.

Tumor-secreted factors are critical regulators of anti-tumor immunity. During cancer progression, malignant, immune, and stromal cells release diverse secreted molecules that collectively form the tumor secretome, which directs the tumor microenvironment (TME) toward immunosensitive or immunosuppressive states by regulating immune cell infiltration, activation, and exhaustion. Immune checkpoint blockade (ICB) therapies, such as anti–PD-1 and anti–CTLA-4 antibodies, have markedly improved survival in select cancers. Despite these successes, most malignancies remain resistant. Prior studies have shown that resistance to ICB is frequently associated with immunosuppressive TMEs driven by extrinsic signals. The tumor secretome therefore represents a promising therapeutic target for overcoming resistance, given its extracellular accessibility and amenability to antibody-based intervention. However, comprehensive and unbiased identification of immune dependencies within the tumor secretome remains a major challenge. Conventional high-throughput pooled genomic screens often fail to capture such dependencies, as surrounding unperturbed cells can compensate for the loss of secreted factors, thereby masking immune-related fitness effects. Our goal is to systematically identify tumor-secreted immune dependencies that act through non–cell-autonomous mechanisms in vivo and to translate these insights into therapeutic strategies. Here, we developed a high-throughput, clonal in vivo CRISPR screening platform. By engrafting single-cell–derived tumor clones in murine lungs and comparing guide representation across immune contexts, we identified genes essential for tumor immune evasion with CRISPR knockout screens. We extended this approach with CRISPR activation-based clonal screens targeting 580 highly expressed genes encoding secreted factors across most human cancer types. These screens were performed in three murine syngeneic cancer models, B16 (melanoma), KP (lung adenocarcinoma), and KPC (PDAC). Using appropriate cell-extrinsic positive controls and cell-intrinsic controls, we demonstrated that clonal screening robustly captures known immunomodulatory factors that are frequently overlooked in mixed-cell pooled screens. Applying this approach, we identified the tumor-associated tissue factor F3 as a previously unrecognized, broadly-acting driver of tumor progression across multiple cancer types. Ongoing studies focus on dissecting the mechanistic basis for the inhibitory role of F3 using spatial profiling of the tumor microenvironment and transcriptional analyses of tumor and immune cells. In parallel, we developed a strategy to selectively augment anti-tumor immunity within tumors. Using cancer-specific fusion transcripts as malignancy markers, we engineered reprogrammable RNA sensing systems (RADARS) that detect these transcripts and trigger expression of immune-stimulatory payloads. Given the inherent limitations of in vivo delivery, these payloads are designed to be secreted, allowing rare intracellular detection events to be amplified into microenvironment-wide immune responses. Together, these studies establish a unified framework for precision immunotherapy that integrates systematic discovery of immune-modulating secreted factors with programmable, tumor-specific immune activation. By identifying secreted immune dependencies that act non-cell-autonomously and pairing them with cancer-specific in vivo delivery designs, this work provides generalizable and tractable strategies to enhance therapeutic selectivity and efficacy across diverse cancer types.

The enteric nervous system is composed of a heterogeneous population of putative sensory neurons, interneurons, secretomotor neurons, and excitatory and inhibitory motor neurons that coordinate intestinal function and immunity. Under inflammatory conditions, specific cytokine receptor signaling in enteric neurons and subsequent neuropeptide action on the immune cells via specific neuropeptide receptors contribute to neuro-immune interactions in the gut. However, the set of all functional cytokine receptors expressed by enteric neurons and the downstream effects of their activation are not fully understood. Owing to this, it is crucial to investigate which specific receptors and signaling cascades are active in different subsets of enteric neurons. In this study, we adopted an in vitro model to differentiate enteric neurons from neurospheres formed from murine neural crest progenitors. We show that enteric neurons exhibit stimulus-specific transcriptional responses to inflammatory cues, including cytokines and bacterial lipopolysaccharide. Targeted gene expression analysis studies on in vitro differentiated enteric neurons by qPCR revealed changes in the gene expression levels of certain neuropeptides, cytokines, and cytokine receptors in response to different stimuli. In addition to this, we were able to identify distinct drivers of neuronal activation in response to inflammation. We also employed immunocytochemistry and fluorescent in situ hybridization-based characterization to study neuronal activation. We further optimized a cFos-GFP system to quantify activated neurons in culture. Together, these findings help us provide a base for building an atlas of enteric neuronal responses to inflammation and use it as a platform to study neuroimmune signaling in ENS.

RNA interference (RNAi) is an antiviral defense mechanism that protects Caenorhabditis elegans against RNA virus infection. MUT-7, a conserved 3′ to 5′ exoribonuclease, has been shown to be involved in RNAi pathway, yet its mechanistic role in antiviral immunity remains incompletely understood. In this study, we investigate the functional contribution of MUT-7 in the context of antiviral defense in C. elegans. Preliminary data in the Kennedy lab demonstrates that MUT-7 exhibits a noncanonical substrate preference, binding double-stranded RNA (dsRNA) and selectively degrading one strand of dsRNA substrates with paired 3′ ends. This activity distinguishes MUT-7 from canonical 3′ exoribonucleases, which typically act on single-stranded RNA with accessible 3′ termini. These findings suggest that MUT-7 may bind and degrade double-stranded viral RNA intermediates generated by viral RdRPs so that MUT-7 can slow or even inhibit viral replication. To assess the role of MUT-7 in antiviral defense, we measured viral load in wild-type, mut-7 loss-of-function, and catalytically dead mutant strains following Orsay virus infection. Loss of MUT-7 results in increased viral RNA levels, consistent with a role in restricting viral replication. However, RNA immunoprecipitation results suggest that MUT-7 doesn’t associate with viral RNAs directly, which might suggest that MUT-7 has either a transient interaction or no interaction with viral RNAs. Another interesting result is that viral load is lower when both MUT-7 and RDE-1 are not present compared to when only RDE-1 is not present, which might suggest that MUT-7 acts upstream of RDE-1 in antiviral defense. Together, our results support that MUT-7 is required for antiviral defense in C. elegans, but the mechanism is still unclear.

Chronic type 2 inflammatory diseases affect millions of people globally. These diseases exhibit tissue remodeling, where inflammatory cells mediate the breakdown of normal extracellular matrix and deposition of excessive submucosal collagen. Tissue remodeling can prompt mechanoreceptor activation through tissue rigidity or integrin activation from binding extracellular matrix proteins. While mast cell activation has a defining role in type 2 inflammation, the presence and functional role of mast cell mechanoreceptor activation is not explored within the literature. We hypothesized that activation of mechanoreceptors on the surface of mast cells could potentiate degranulation and/or mediator release, thereby contributing to type 2 inflammatory disease. We generated mast cells from circulating progenitors in peripheral human blood, evaluated the expression of diverse mechanoreceptors (mechanosensitive ion channels and integrins) on mast cell surfaces, investigated mechanoreceptor upregulation following cytokine treatment, and demonstrated the capacity for mechanoreceptors to affect mast cell activation. We have found diverse mechanosensor expression on human mast cells, with differential expression depending on mast cell subtype and a stable phenotype unaffected by cytokine treatment. Contrary to our original hypothesis, we found variable effects of mechanosensor stimulation on mast cell activation, with one significant finding in TRPV4 activation attenuating mast cell degranulation and mediator production; however, we have also discovered a potential new role of integrin-mediated lipid mediator production independent of degranulation. Thus, we present our findings of novel perspectives on mast cell functionality via mechanosensing.