Brain extracellular matrix (ECM) structure mediates many aspects of neuronal function. When ECM structure becomes dysregulated in neurological disease, one resulting impact is impaired neuronal function. Therefore, probing changes in the ECM structure could provide insights into disease mechanisms and expose potential therapeutic pathways. Previous work in our group determined that degrading neural ECM structures leads to a significant increase in the diffusive ability of nanoparticles navigating the brain extracellular space. However, this diffusion-based analysis provides little insight into changes in ECM-specific morphology or structure; analysis only predicts if they are present and the degree of alteration from normal. With this project, we aimed to characterize changes in perineuronal net (PNN) structure with high spatial resolution using a fluorescence-based imaging approach. PNNs, a structure that impact neuronal function were stained using a fluorescently labeled lectin (Wisteria floribunda agglutinin) and images acquired via confocal microscopy. Images were collected from the cortex of brains spanning an age range of post-natal day 14 to adulthood. We first manually quantified morphological features associated with PNNs, including the total number of branches, average branch length, and total branch length using the image processing program ImageJ. To reduce image processing time and minimize user-bias, I am building a Python-based, automated quantification workflow for future use. Regarding the manually quantified PNN features, we observed an increase in both the total branch length/PNN and average branch length/PNN as brains increased in age. The total number of branches/PNN remained relatively consistent across age groups. Future work will focus on applying the same approach to a model of neonatal hypoxia ischemia to study injury effect on PNN structure. Collectively, this project established a methodology that can be applied for enhanced characterization of ECM-related structural changes induced by neurological disease, and has the potential to unveil new avenues of therapeutic intervention.

HSV-2 is a sexually transmitted disease of global importance, with an estimated infected global population of over 500 million people, as estimated by the WHO. HSV-2 is a lifelong infection which results in painful, recurrent lesions in the genital area. Previous research has shown that high numbers of regulatory T cells (suppressive immune cells important for maintaining tolerance) are present in the mucosal tissue alongside traditional CD4+ and CD8+ T cells (T cells important for an adaptive immune response), and their role is not fully understood. The reporter strain “Nedel” is a strain of HSV-2 with an inserted mNeonGreen fluorescent marker, allowing us to visualize HSV-2 infection. Our current project is to characterize the viral kinetics of the reporter strain of HSV-2 and compare it with WT HSV-2, as well as visualize the virus in infected cells via immunofluorescence and flow cytometry. We have infected mice intravaginally with both strains of virus, collected vaginal washes for plaque assays (to determine viral load) and harvested their vaginal tracts for flow cytometry. Our first round of experiments have shown that while the general frequency of immune cells were comparable, there was significantly lower activation of these cells in the Nedel-infected mice as compared with the WT-infected mice. In ongoing experiments, we have infected the mice with a higher dose of Nedel virus in an effort to make infection more comparable to WT, and we will be harvesting vaginal tracts for flow cytometry analysis to see whether we can visualize the mNeonGreen. Our overall goal is to utilize the Nedel HSV-2 strain to investigate the role of regulatory T cells in modulating anti-viral immune response, with a broader goal of finding treatments and an effective vaccine for HSV-2.

Breast milk is essential to the health and development of a child, containing antibodies that protect infants from common illnesses. However, exclusive breastfeeding is not always possible, and no infant formula substitutes for maternal antibodies. Previous studies on mice showed high germinal center (GC) B cell levels in response to the absence of maternal antibodies from breast milk early in life. Germinal centers B cells are involved in the adaptive immune system by secreting high affinity antibodies. However, little is known about the consequence of this, making characterizing the isotype, location, and duration of the antibody response in neonates (newborns) lacking maternal breast milk antibodies essential. For this project, I designed and optimized a tissue preparation and flow cytometry panel to assess memory B cells, GC B cells, and plasma cells (cells that secrete antibodies to fight infections and disease). The flow panel uses the cell markers CD138 and B220 to identify plasma cells by isolating the cells that are positive for CD138 and negative for B220. However, the marker CD138 can be sensitive to collagenase, resulting in the potential failure to identify plasma cells successfully. For this reason, I tested a range of collagenases, including Collagenase A, D, and IV. Concluding that CD138 was being cleaved off by all tested collagenases, I then used TACI as a new type of plasma cell differentiation marker. I evaluated these protocols by the viability of cells and the plasma cells' frequency. This protocol allows for the determination of localization, persistence, and isotype of early life B cells activated in the absence of breast milk.

The body’s immune system utilizes T cells to identify and kill tumor cells of growing cancers. However, hematological and solid tumors inhibit T cells by producing a tumor microenvironment (TME) that presents inhibitory and death signals that hinder the T cells cancer fighting potential. At Dr. Oda’s lab we work to overcome this TME obstacle by engineering exogenous proteins that can be expressed in T cells. These immunomodulatory fusion proteins (IFPs) combine an inhibitory ectodomain with a costimulatory endodomain. Therefore, when these novel T cells receive deadly messages from tumors, they instead interpret and rewire the signal in ways that increase their cancer combatting abilities. We have had success in engineering Fas-4-1BB and CD28/CD200R IFPs data supports the IFP’s ability to increase proliferation, metabolism, and antitumor properties along with decreasing exhaustion in T cells. This presentation will focus on our recent investigations into an IFP that modulates the relationship between SIRPa and CD47. It is known that tumor cells express CD47 which binds to SIRPa on macrophages and gives an anti-phagocytic “don’t eat me” signal. CD47 also binds to SIRPa on dendritic cells and shuts them down, effectively inhibiting an antigen presenting cell that is key in communicating cancer antigens to T cells for identification. By combining the SIRPa ectodomain with the costimulatory CD28 endodomain, we can manipulate this inhibition by CD47 which is overexpressed in TMEs. I use gateway cloning to prepare vectors for transfection. I transfect SIRPa/CD28 constructs into PlatE viral producing cells and transduce the virus into T cells for expression. From here, I set up serial killing over time assays to collect and analyze in vitro data. This research on IFPs can lead to improved clinical treatment for cancer patients, resulting in increased survival rates across a range of different cancers and patients of all ages.

 Chronic Mucocutaneous Candidiasis (CMC) is a recurring Candida infection that occurs in patients with T-cell deficiencies, and specifically infects skin, nails, and mucous membranes (Okada, Satoshi et al, Clinical & translational immunology, 2016). Signal Transducer and Activator of Transcription 1 (or STAT1) is a gene known for its role in the IL-17 signaling pathway, which functions in immune response; this specific pathway is critical to fighting off infections such as Candidiasis . Previous studies have shown that autosomal dominant mutations in STAT1 on exon 11 can lead to chronic Candida infections . We have identified a case of CMC which we suspect are caused by gain-of-function mutations in the STAT1 gene. To confirm this suspicion, we performed genetic testing of archival tissue samples. We designed primers targeting exon 11 of the STAT1 gene, and conducted polymerase chain reactions followed by Sanger sequencing. I will be working with the team to anayze the samples and determine whether or not STAT1 mutations are present in these patients, and if these are novel mutations. Identification of STAT1 mutations will be paramount to further understanding the genetic background of patients with CMC, and informing future studies to potentially target the IL-17 pathway in treating CMC.

CD8+ T-cell exhaustion during chronic human immunodeficiency virus (HIV) infection is characterized by increasing expression of inhibitory markers on the cell surface that lead to decreased effector function and dampened immune responses that are essential to achieving therapeutic vaccine efficacy. Inhibitory marker PD-1 is upregulated on exhausted CD8+ T-cells, and blockade with a monoclonal antibody (aPD-1) can help reverse exhaustion. We hypothesized that dosing with aPD-1 would boost the immune system and decrease expression of other exhaustion markers throughout infection to improve therapeutic vaccine responses. To investigate this hypothesis, we studied how exhaustion progresses over time in simian-human immunodeficiency virus (SHIV)-infected Rhesus macaques treated with a novel combinatorial therapeutic regimen consisting of a conserved-elements vaccine to circumvent viral mutants, GS986 to reverse latency, CCR5 gene editing to prevent viral entry, and aPD-1 to reverse CD8+ T-cell exhaustion. We characterized exhaustion in peripheral blood mononuclear cells and mesenteric lymph nodes with surface staining and flow cytometry, with a focus on exhaustion markers PD-1, TIGIT, CTLA-4, LAG-3, and TIM-3 at various timepoints throughout infection and vaccination. Although we observed no differences in viral burden between treatment groups, we observed higher CD8+ T-cell vaccine responses in animals receiving aPD-1 compared to vaccinated animals that did not receive aPD-1, suggesting aPD-1 improved vaccine responses. Although we did not find a correlation between PD-1 signaling and any exhaustion markers, we observed a significant negative correlation between CD8+ T-cell vaccine responses and pre-vaccination TIGIT levels (Spearman r= -0.75, p=0.007). We also found that PD-1 and TIGIT are largely independently expressed on CD8+ T-cells. Taken together, these data suggest a role for dual blockade of PD-1 and TIGIT to improve vaccine efficacy in future studies. Defining the impacts of CD8+ T-cell exhaustion on therapeutic vaccine immunogenicity is crucial to improving combinatorial immunotherapy towards a cure for HIV.