T cells are a crucial part of the human immune system that play a role in the response to diseases from type 1 diabetes to COVID-19. All T cells undergo genomic recombination to create distinct T cell receptor complex (TCR) cell surface molecules that recognize antigens presented by major histocompatibility complex (MHC) molecules, thereby triggering a series of signaling pathways that culminate in T cell activation and cell division. Afterwards, daughter progeny share the same unique TCR molecules. In my project, I will be examining the role TCRs shared within a single individual (private) and between individuals (public) during COVID-19 infection. While the existence of these classes of TCRs is well documented, their functional roles remain unclear. My hypothesis is that the presence of public TCRs is linked to the strength of an immune response and, hence, to different disease severity in humans. We reason that common evolution may have led to the rise of these public TCRs because of their major role in the human body’s antiviral immune response. This hypothesis predicts reduced levels of public TCRs results in an inability to fight disease, whereas high levels of public TCRs will be associated with less severe disease. To test this, I will use a mixture of single cell RNA analysis and statistical tests on a dataset of TCRs acquired from blood samples of 254 SARS-CoV-2 patients and 16 healthy patients. Using a multiomic approach will allow me to also find genetic links between cells of interest. The data includes baseline and acute readings for each patient, and can be subsetted to cells with expanded TRA and TRB junctions individually. This experiment will help advance our knowledge about the function of public TCR chains and their role in fighting disease. Furthermore, our data could have translational applications for disease biomarkers.

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 study on Digital Spacing Profiling in Antibody-Mediated rejection (AMR) looks at patients post-kidney transplant. This study involves the use of Digital Spacing technology to analyze the tissue samples of patients suffering from antibody-mediated rejection. Antibody-mediated rejection remains the most formidable cause of kidney transplant failure. Digital spatial profiling of biopsy specimens is a novel technology that provides unprecedented insight into spatially registered molecular changes to better define pathophysiology and potential therapeutic targets. We used the Nanostrong GeoMx platform to define the spatial transcriptome of AMR and compared these results with pathologic, laboratory, and clinical data. We hope that the results from this study will provide novel insight into the pathophysiology of AMR, and lead to improved diagnosis and treatment of AMR. The primary goal is to characterize patients and kidney transplant genomic profiles using digital spatial profiling in the setting of biopsy-proven antibody-mediated rejection, de novo donor-specific antibodies, and/or donor-derived cell-free DNA. Rejection is caused by the body’s response to the transplanted tissue and is typically treated with medication. This study looks at patients who may have different reasons to receive a kidney transplant and how their treatment impacts their organ rejection. The study utilizes clinical and pathological data to learn more about antibody-mediated rejection in kidney transplant recipients, specifically. With IRB approval, we identified eligible subjects through the review of UWMC electronic health records, abstracted patient data, then looked at the patterns from the profiling. Data analysis is underway. This work provides an atlas for the future prevention of AMR, research on this topic is essential to understanding and helping transplant patients live the healthy lives that transplants promise them. Along with that, this research furthers the medical field's scientific understanding of the human body and how to create more effective treatments for illnesses.

Merkel cell carcinoma (MCC) is a rare and aggressive skin cancer with a mortality rate of ~30%. In ~80% of cases, MCC development is attributed to the integration of Merkel cell polyomavirus (MCPyV) DNA into the host’s genome, leading to the expression of viral oncoproteins and tumorigenesis. Developing treatments that sustain immunity against MCC is imperative to address recurrent and/or progressive disease. In many cancers, tumor-infiltrating B cells have been associated with better prognosis and response to immunotherapies. However, the mechanisms by which B cells contribute to tumor immunity in humans have been difficult to resolve in part due to the inter-patient heterogeneity of tumor-specific antigens. The shared nature of MCPyV tumor antigens in MCC allows for MCC-specific B cell responses to be studied across patients. Using DNA-barcoded and fluorescently labeled viral oncoprotein tetramers, we analyzed the transcriptome, proteome, and receptor repertoire of MCC tumor-infiltrating B cells in 12 patient samples at single-cell resolution. From paired heavy and light chain sequences, we cloned 8 antibodies from B cells specific for the MCPyV oncoproteins to confirm binding to MCC-specific antigens. Transcriptomic and proteomic analyses of MCPyV-specific B cells revealed heterogeneity of intra-tumoral B cell responses. Interestingly, we found that the absence of MCC-specific germinal center (GC) B cells in MCC tumors associates with disease progression: ~80% of patients with no detectable GC B cells had MCC progression within a year post-surgery, whereas patients with detectable GC B cells remained progression-free a year after surgery (n=12, p=0.0043). These results suggest strong synergy between B cells and T cells may regulate tumor growth, as B cells rely on signals presented by T cells to differentiate into GC cells. Our long-term objective is to identify B cell phenotypes associated with anti-MCC responses to develop therapeutics that boost cancer-specific immunity.

Soluble factor signaling between immune cells and fibroblasts is critical in regulating biological processes. However, it is often dysregulated in diseases and leads to physiological changes, including airway inflammation in asthma and allergies. One immune cell type that can be attributed to airway inflammation is eosinophils (EOS). When activated by interleukin-3 and heat-aggregated immunoglobulin G, EOS release certain soluble factors associated with the activation of lung fibroblasts. To investigate the interactions between human lung fibroblasts (HLFs) and EOS, we used the open microfluidic coculture device. This device has two chambers, in which two types of cells can be cocultured in the shared media while being physically separated by a half wall. We found that HLFs in coculture with activated EOS had the highest levels of proinflammatory gene expressions and proinflammatory cytokines. However, the exact mediators responsible for promoting these biological processes are still uncertain. We hypothesize that EOS secrete a cytokine, interleukin-1 alpha (IL-1a), and a protein, transforming growth factor alpha (TGFa), to be consumed by HLFs, triggering proinflammatory responses of HLFs. The goal of this study was to elucidate the roles of IL-1a and TGFa in airway inflammation. HLF-EOS cocultures are seeded in the microfluidic coculture device, then IL-1a, TGFa, and their respective cellular receptors are neutralized using antibodies. Enzyme-linked immunosorbent assays are used to measure the level of EOS-derived neurotoxins after their activation. Then, reverse transcription quantitative-polymerase chain reactions are used to quantify gene expression levels relevant to proinflammatory responses of HLFs, in addition to multiplex immunoassays to analyze the secreted soluble factors from both cell types. We anticipate that HLF-EOS cocultures treated with neutralizing antibodies have lower expression levels of proinflammatory genes than cocultures without antibodies. Findings from this study will help us better understand the key regulators that promote proinflammatory behaviors of HLFs in airway inflammation.

Development of mammalian immune systems begins early in gestation and may be influenced by maternal microchimeric cells (MMc) which are passed from mother to fetus. The largest number of MMc are antigen-experienced T cells, however, the timing of cell transfer into the fetus is unknown. We hypothesize that maternal infection and vaccination will result in an increase in antigen-specific T cell clones in the mother and that tracking these clones into the fetus will allow us to predict the timing of MMc transfer. In order to define the frequency and timing of these maternal immune stimuli, I compiled first trimester health survey responses from a prospective cohort study of pregnant people to analyze acute maternal infection and vaccination events. In the 20 women thus far enrolled during fall 2022 and followed through first trimester, three women reported respiratory illness and three women reported gastrinestinal illness; no women reported influenza or COVID-19 infection. Eleven women reported influenza vaccine receipt and eight reported bivalent COVID-19 vaccine receipt. These data indicate that infection and vaccination occur frequently in first trimester pregnant women. This high rate of immune stimuli may reflect the fall season of enrollment, when a high number of viruses were circulating in the community and both influenza and bivalent COVID-19 vacines were recommended for pregnant women. We will next sequence the T cell repertoire of women around these events to determine whether we can identify T cell clones that “time-stamp” the repertoire at different gestational ages. Understanding the timing of MMc transfer across the placenta will enable future work to manipulate the maternal T cell compartment and maximize the transfer of antigen-specific MMc T cells to the fetus. This has particularly important implications for global communities that face a disproportionately large burden of infectious diseases.