This abstract has been withheld from publication.

Many T-cell-driven autoimmune diseases are incurable and also biased to the female sex. 80% of autoimmune disease cases are in females.This suggests that sex-specific processes may contribute to autoimmune disease pathogenesis through altered T cells. X-inactive specific transcript (Xist) is a long noncoding RNA that epigenetically silences X chromosomes when two or more X chromosomes are present. Recent literature suggests that Xist within immune cells may impact regulation of X-linked immune related genes. However, Xist’s impact on T cell phenotype and subsequent role in autoimmune disease development is not well characterized. In order to explore the role of Xist, we used CRISPR, a gene editing tool that is able to make precise cuts in the genome using a guide RNA, to knock out Xist in CD4+ T cells. Then, we evaluated changes in X-linked immune gene expression through rt-qPCR such as Sh2d1a and Firre. We saw that there was a decrease in expression of Xist through CRISPR editing but no change in expression in Sh2d1a or Firre. Through further exploration of these immune genes, we can better understand what causes autoimmune diseases to be biased towards the female sex. Additionally, we hope to better characterize the underlying causes for autoimmune diseases in order to work towards guiding new treatments since autoimmune diseases are often incurable.

Aging is marked by a progressive decline in physiological function, driven in part by the accumulation of senescent cells in aging tissues, which are characterized by their irreversible cell-cycle arrested state and capacity to secrete inflammatory molecules. Among cell types that are susceptible to this fate, senescent macrophages have emerged as key contributors to chronic inflammation and drivers of age-related pathologies. Our lab identified Cytidine Monophosphate Kinase 2 (CMPK2), a mitochondrial enzyme key for forming nucleotide precursors for mitochondrial DNA (mtDNA) synthesis, as a potential regulator of this inflammatory program. We observed that Cmpk2 expression was upregulated in aged and senescent macrophages, coinciding with heightened levels of mtDNA, a known trigger of sterile inflammation independent of infection. To address this, CRISPR-mediated Cmpk2 knockout senescent macrophages were generated, which resulted in a significant reduction of mtDNA abundance, sterile inflammation mediated by type-1 interferons, and downstream activation of interferon-stimulated genes. Together, these findings support CMPK2 as a central regulator linking mitochondrial nucleotide metabolism to cytosolic DNA-detecting interferon signaling in senescent macrophages. By uncovering this connection, our work positions CMPK2 as a promising therapeutic target to attenuate macrophage-driven chronic inflammation and promote healthy immune aging.

Adjuvant-induced innate immune activation is needed for a robust immune response, which is crucial for immunotherapy strategies such as cancer vaccination. Commonly used adjuvant polyinosinic:polycytidylic acid (poly(I:C)), shuts down translation of mRNA, thereby rendering poly(I:C) inefficient to use with the mRNA vaccine platform. Since antigen-presenting cells must be activated and present antigen to T cells, we focused on generating innate immune activation while maintaining sufficient antigen expression. We hypothesized that using a novel RNA lipid nanoparticle (RNA-LNP) could achieve innate immune activation while maintaining mRNA antigen translation. To investigate the effect of our RNA-LNP adjuvant, we transfected bone marrow-derived macrophages (BMDMs) and assessed translation levels via Western blot. We also investigated innate immune activation by qPCR to compare the immunostimulatory properties of our RNA-LNP adjuvant compared to poly(I:C). Our RNA-LNP molecule efficiently generated cytokine induction in a dose-dependent manner, as shown in the qPCR. Poly(I:C) illustrated a dose-dependent increase in phosphorylation in the protein kinase R pathway, indicating antiviral translation suppression, but our RNA-LNP adjuvant preserved protein translation across concentrations. By characterizing and optimizing our RNA-LNP, we can induce high antigen expression while stimulating an innate immune response to provide a potential cancer therapeutic.

Immune checkpoint inhibitor (ICI) cancer therapy has significantly improved cancer patient outcomes by activating immune cells to mediate tumor destruction. Combination ICI therapies (e.g. anti-PD1 + anti-CTLA4) elicit greater anti-tumor responses compared to single agent ICI, but are also associated with a higher risk of immune related adverse events (IrAEs) that lead to treatment discontinuation, hospitalization, and death. One form of IrAEs, ICI-Thyroiditis, involves autoimmune attack of the thyroid. To understand why combination ICI leads to increased IrAEs, our lab used single-cell RNA sequencing to analyze data from patients with ICI-Thyroiditis. We found that patients who received combination ICI therapy had increased differentiation of CD4+ T cells to T follicular helper (Tfh) and T peripheral helper (Tph) cells compared to patients who received anti-PD1 alone. This was accompanied by increased germinal center B cells, which altogether points to the development of tertiary lymphoid structures (TLS) in the thyroids of patients treated with combination ICI. This finding was validated in our mouse model of IrAEs, where we found that combination ICI increased the frequency of Tfh, Tph, and B cells in the thyroid-draining lymph node by flow cytometry and formation of TLS in the thyroid by immunofluorescence. In summary, our data demonstrates that an upregulation of CD4+ Tfh/Tph and B cells due to combination ICI are associated with formation of TLS in the thyroid and contribute to an increased rate of ICI-Thyroiditis.