Bronchoscopy with bronchoalveolar lavage (BAL) is a common approach to assess the graft in lung transplant recipients. However, bronchoscopy is an invasive approach that carries procedural risks. Identifying plasma biomarkers levels which correlate with those measured in BAL fluid enables less invasive investigations. This study aims to determine the correlation between biomarkers measured from plasma and BAL fluid. We analyze paired plasma and BALF (BAL fluid) samples collected from lung transplant recipients undergoing for-cause bronchoscopy (n = 95 individuals and inclusive of 164 encounters). Researchers collect paired samples from each participant within a two-hour timeframe. Researchers measure a panel of 39 biomarkers in samples using electrochemiluminescence assays. We evaluate plasma/BAL correlation data using Pearson correlation coefficients on log-transformed concentrations. Among participants, the average age is 61 years, 74% are male, 93% are white, with Restrictive lung disease (60%) being the most common pre-transplant pulmonary disease. The median time from transplant to bronchoscopy is 412 days (IQR: 194-741). Results indicate that correlational data is highly variable. The two most highly correlated biomarkers are IL-12/IL-23p40 (r = 0.67) and CXCL10 (r = 0.64). There are no biomarkers that have significant inverse correlations. The levels of 11 out of the 39 measured biomarkers show moderate correlation, ranging from r = 0.20 to r = 0.45. Many of these biomarkers are chemokines related to immune cell migration to the lung (e.g., CCL2, CCL3, CCL4, and CCL22). A key implication of this finding is that researchers should exercise caution when extrapolating alveolar biology from circulating (plasma) samples. Future analyses will test whether the ratio between paired BALF and plasma biomarker levels provides biological or clinical insight beyond using these biomarkers measured in isolation.

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.

Merkel cell carcinoma (MCC), a rare skin cancer, is mostly driven by integration of the Merkel cell polyomavirus which encodes T-antigen (T-Ag) proteins. Previous research has shown that T & B cells target T-Ag. Indeed, patients with virus-driven MCC produce T-Ag-specific antibodies that are useful to track disease progression. These antibodies do not play a direct role in MCC immunity as T-Ag proteins are intracellular. Our group has recently found that in tumors, T-Ag-specific B cells with germinal center or antibody-secreting phenotypes strongly predict improved MCC outcomes. These intratumoral B cell phenotypes reflect a robust cancer-specific T cell response. In contrast, T-Ag-specific B cells in the blood of MCC patients are predicted to predominantly have a memory or naive phenotype, and it is unknown if they contribute to anti-tumor immunity. We used fluorescently labeled T-Ag-proteins and flow cytometry to assess B cell responses in blood at the time of MCC diagnosis. In total, we analyzed samples from 23 patients whose MCC recurred within 3 years of diagnosis and 24 samples from stage- and age-matched MCC patients whose disease did not recur. We found no difference in the frequency of all circulating B cells (regardless of T-Ag-specificity) between patients who did and did not develop MCC recurrence. In contrast, higher frequencies of total memory B cells (CD27+IgD-IgM-) were associated with an increased risk of disease recurrence (HR 3.67 [1.58- 8.55], p=0.003). Intriguingly, T-Ag-specific memory B cells were also more abundant in the blood of patients who ultimately developed MCC recurrence (HR 2.82 [1.22- 6.53], p=0.012). Together, our results demonstrate that higher frequencies of circulating memory B cells associate with worse MCC outcomes. These findings suggest that the functional state of total and T-Ag-specific circulating B cells reflect their immune response within MCC tumors.

Preterm birth is a leading cause of neonatal morbidity and mortality, with intra-amniotic infection and inflammation being major contributors to early preterm labor (PTL). Despite ongoing research aimed at reducing inflammation in neonates, most studies have focused on post-delivery while few have been done prior to delivery. IL-1 is a central upstream mediator of inflammation in the amniotic cavity and the neonate. IL-1 is a key cytokine that is responsible for induction or propagation of the cytokine cascade responsible for PTL. Rytvela, an interleukin-1 receptor antagonist made up of seven D-amino acids, acts as a selective antagonist of IL-1 signaling, which could be used to act as a therapeutic approach to reduce inflammation and prevent PTL. The purpose of this experiment is to determine if interleukin-1 (IL-1) is a key molecular target for the development of antenatal therapeutics to prevent PTL and fetal injury. We hypothesize that Rytvela administered intravenously to the mother will cross the placenta and be detectable in the amniotic fluid and fetal plasma, suggesting that Rytvela could effectively block IL-1 signaling in the fetus and therefore reduce fetal inflammation. Maternal blood plasma samples were drawn at Day 1, 2, 6 and 10 post infusion. To confirm the transfer of Rytvela to the fetus, we used liquid chromatography-mass spectrometry (LC-MS) to detect the drug, looking at integration, peak identification, and backlog pressures to see if Rytvela is detectable in maternal plasma. Rytvela was detected and luminex plates were run to measure cytokine levels. After GBS infection, Il-1 beta and Il-23 concentrations increased. After Rytvela administration, the concentration of the pro-inflammatory cytokines decreased. Future directions will involve measuring cytokine levels at these time points and correlating them with Rytvela infusion to evaluate the drug’s impact on maternal-fetal inflammation

Copy number variation (CNV) is associated with genetic disorders in humans. One particular kind of CNV is a gene triplication in which the central copy is inverted. How such structures arise is poorly understood but is of great interest because of their association with cancer and other genetic disorders. We find similar amplified structures in budding yeast Saccharomyces cerevisiae, and therefore, we can use yeast to understand the mechanism that generates them. The Brewer and Dunham labs have proposed Origin Dependent Inverted Repeat Amplification (ODIRA) as a model that explains inverted CNVs. To understand which proteins/enzymes contribute to amplification, we are implementing gene deletions to observe the effect on the production of these ODIRA amplification events. ODIRA events are rare and thus large sample sizes are required to detect them. To streamline their identification, I am developing a system that uses color as a visual indicator of CNV. When a single copy of the bacterial gene, VioA, is expressed in yeast it produces light purple colonies; in multiple copies, the colonies are a darker shade of purple. I am inserting a single copy of the VioA gene into a region of the yeast genome that undergoes inverted triplication events. Simply scanning plates for dark purple colonies will enable me to screen for ODIRA events quickly and measure their frequency. Using this visual indicator will allow me to rapidly screen different yeast mutants and determine the role they play in the ODIRA amplification mechanism.

Maintaining the integrity of genetic material and preventing changes over time is essential for normal cellular function. This genomic stability is directly affected by the DNA replication process. Replication must be both accurate and efficient; mutations that affect DNA replication can cause genomic instability and changes in the genetic makeup of the cell. Through a genomic instability screen in mice, researchers discovered a single base pair mutation in a highly conserved gene required for unwinding DNA during DNA replication. The presence of this single base pair substitution, called Chaos3, in both copies of the gene causes female mice to develop mammary tumors. We have found that in the yeast Saccharomyces cerevisiae, the corresponding Chaos3 mutation decreases activation or “firing” of some replication origins—the sites where DNA replication begins. Chaos3 does not affect all early firing origins in the genome; rather, origins near centromeres are specifically affected, thereby delaying replication of those centromeres, causing chromosome loss. We found that when an affected origin is replaced with an unaffected one, firing levels are restored to wild type function and that chromosome loss is rescued. To further understand what components are essential for timely DNA replication, and why only a subset of origin sequences are sensitive to the Chaos3 allele, I am focusing on the origin sequences directly. I am mutating the origin sequence itself and separately deleting different genes whose products have potential interactions with origin sequences. My research aims to advance the understanding of the role these genes play in the activation of origins for timely DNA replication and how the Chaos3 mutation may be interrupting normal function of these processes. This knowledge can help identify key molecular mechanisms that drive cancer development in higher eukaryotes.