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James Rubenstein , Department of Medicine; University of California, San Francisco

Characteristics of PCNSL and its Environment

Abstract:

Lymphoma is the cancer of the lymphocytes (B-Cells and T-Cells) that constitutes of nearly 4% of the diagnosed cancers. Within, lymphomas, there are two major division: Hodgkin's Lymphomas and Non-Hodgkin’s Lymphomas (NHLs). NHLs constitute of nearly 5% of the head and neck malignancies, and occur with a variety of phenotypes. One specific type of NHL that occurs in 30% of the NHL population is Diffuse Large B-Cell Lymphoma (DLBCL). Nodal DLBCL is generally treated using the R-CHOP immunotherapy (Rituximab, Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone). However, these drugs have shown to not penetrate the Blood-Brain Barrier efficiently in cases of Primary Central Nervous System Lymphoma (PCNSL). Usually, PCNSL is treated using methotrexate, which has a failure rate of 35%, and patients have methotrexate resistance. 

The Rubenstein Lab is focused on creating novel immunotherapies for patients who show methotrexate resistance. In this regard, two aspects of the PCNSL were studied: (1) MHC Expression on the lymphomas and (2) macrophages in the tumor microenvironment. 

To study the expression of MHC I, we studied the expression of TAP-1. Strong correlation between patient survival (both progression-free survival and overall-survival) and TAP-1 expression. Patients with strong TAP-1 expression responded better to methotrexate treatment and did not relapse. Patients with mutated TAP-1 and/or lower TAP-1 expression did not respond well to methotrexate treatment and relapsed.

Previously, the Rubenstein Lab discovered a strong correlation between the presence of macrophages in the tumor microenvironment and patient survival. Specifically, patients with M1 macrophage phenotype had a better survival. As an extension, we wanted to better understand the role of M1 macrophages in the tumor microenvironment. Here, we found that the macrophages used TLR7/8 signaling to convert to M1 phenotype and produce Granzyme B.

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Greg Barton , Department of Molecular & Cell Biology, University of California, Berkeley

Investigating the Synergistic Anti-tumor Effect of Akkermansia muciniphila and Immune Checkpoint Inhibitor

Abstract:

The gut microbiota plays a salient role in mediating host physiology and immunity. Several studies link immune checkpoint inhibitor (ICI) efficacy in extraintestinal cancers to gut microbiota composition, with commensals, such as Akkermansia muciniphila (Akk). Recently, studies had found that Akk treatment during anti-PD1 immunotherapy showed potential clinical relevance; however, the differential effects on antitumor responses by the different strains of Akk are not yet elucidated. In this study, whether specific pathogen-free mice and gnotobiotic mice colonized with representative Akk strains exhibit differential anti-tumor responses during ICI therapies is examined.

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Laurent Coscoy , Department of Molecular & Cell Biology

Determining the role of the Virally-Encoded microRNAs in ERAAP Downregulation During Mouse Cytomegalovirus Immune Evasion

Abstract:

Murine cytomegalovirus (MCMV) is a betaherpesvirus often used as a research model for investigating viral pathogenesis in humans. Upon recognition by host machinery, viral peptides are processed by the proteasome and are transported to the endoplasmic reticulum (ER), where they are trimmed to appropriate lengths by ER aminopeptidases (ERAAP) and presented by MHC Class I. The MHC Class I pathway is essential for cytotoxic T lymphocytes (CTLs) to induce programmed cell death in infected cells. However, MCMV has evolved several strategies to bypass the host’s immune response, such as downregulation of the ERAAP protein. As a result, loss of ERAAP function make MCMV-infected cells less susceptible to apoptosis by CTLs, allowing the virus to replicate and infect neighboring cells.

One possible mechanism is that MCMV could encode viral microRNAs that inhibit ERAAP translation. In bioinformatic analyses, several MCMV-encoded microRNAs have been predicted to bind with the noncoding regions of ERAAP mRNA and lead to its downregulation. When infecting an ERAAP-expressing vector that contains different noncoding regions than endogenous mRNA, no ERAAP downregulation was observed. This suggests that that the 3’ UTR or 5’ UTR of ERAAP may interact with MCMV. By identifying one or more candidate microRNAs that could mediate ERAAP downregulation in infected cells, these discoveries could provide potential targets for the development of antiviral treatments against cytomegalovirus infection.

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Igor Grigoriev , Lawrence Berkeley National Laboratory

Conserved Algal Plastid Genes

Abstract:

Algae are eukaryotes that gained photosynthetic capabilities by acquiring plastids through endosymbiosis. While algal plastids originate from a single primary endosymbiosis, they have a complex history that resulted in two distinct yet related groups— green plastid and red plastid algae. Algae are a diverse and complex group, responsible for about half of all photosynthesis on earth. Looking at 62 algal plastid genomes, including 24 greens and 38 reds, we identified genes conserved across different groups to create a list that can be used to evaluate plastid sequence completeness. Then, we analyzed trends in conserved genes and explored the fate of lost genes using the Mamiellophyceae clade. 

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David Raulet , Department of Molecular & Cell Biology

High IL-2 Concentrations in vitro Drive Decline in Natural Killer Cell Degranulation and Cytotoxicity

Abstract:

Natural Killer (NK) cells play a critical role in the innate immunity against pathogens and the development of cancer. The function of NK cells depends on the intricate balance of activating and inhibitory receptors on its cell surface. When the stimulating signals overweigh the inhibitory signals, NK cells are licensed to kill. However, previous work has uncovered that when NK cells are engaged in vitro with continuous positive stimulation, there is a desensitization effect resulting in reduced functionality. In this project, the impacts of IL-2, an important cytokine for NK cell function, are investigated for its role in NK cell desensitization. When looking at NK functionality under different IL-2 concentrations, initial experiments showed that at higher IL-2 concentrations (2000 IU/mL), there is greater NK cell proliferation. We further demonstrated that the NK cell’s ability to degranulate, measured by CD107a levels, following in vitro stimulation using  ɑNK1.1 antibody declines as IL-2 concentration increases. Additionally, we found that the observed NK desensitization is not attributable to the presence of CD4 T cells, CD8 T cells, regulatory T cells (Tregs), or B cells as the depletion or elimination of these cells did not restore NK functionality. The decline in NK cell degranulation was also seen to have further impacts on NK cytotoxicity and specific lysis of RMA-B2m-/- cells. NK cells cultured at a high IL-2 concentration (2000 IU/mL) had the lowest cytotoxicity compared to its  counterparts cultured in lower concentrations. Overall, these results provide insights into NK desensitization and have future implications on the administration of IL-2 as a cancer immunotherapy.

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Dr. Suzanne Fleiszig, Dr. David Evans , UC Berkeley School of Optometry

Contact Lens-Induced Corneal Para-Inflammation: Mechanisms of Persistence After Lens Removal

Abstract:

Purpose:

Contact lens (CL) wear triggers para-inflammation in human and mouse corneas. Previous investigations in mice showed that para-inflammation involves a significant increase in Lyz2+ myeloid-derived immune cells after 24h of lens wear, while the cornea remained free of visible pathology and maintained optical clarity. Here, we studied persistence of this lens-induced para-inflammatory response after lens removal.

 Methods:

LysMcre mice were used to visualize corneal Lyz2+ cells. One eye of each mouse was fitted with a custom-designed mouse contact lens and contralateral eyes were used as no-lens wear controls. The following conditions were tested: 24h CL wear, 48h CL wear, 24h CL wear+24h CL removal, and no lens wear. After euthanasia, the eyes were enucleated, fixed them in 2% paraformaldehyde, and used confocal imaging to quantify corneal Lyz2+ cells. qPCR was used to evaluate gene expression of pro-inflammatory cytokines IL-1β, IL-18, TNF-α, IL-17A, IL-17F, RORγt, and IL-23. Two-way ANOVA was used for statistical analysis with P<0.05 considered significant.

 Results:

After 24h and 48h of lens wear, corneas showed an elevation of ~1.2-fold and ~1-fold respectively in Lyz2+ cells compared to contralateral controls, while maintaining optical clarity and showing no clinical evidence of inflammation. However, 24h after lens discontinuation, corneas showed a 48% increase in Lyz2+ cells compared to 24h of lens wear alone. No significant difference was observed between 24h and 48h of lens wear. Gene expression analysis showed upregulation IL-17F and RORγt after 24h and 48h of lens wear, but significant downregulation of these genes at 24h after lens discontinuation.

 Conclusion:

Contact lens-induced para-inflammation in the murine cornea involving Lyz2+ cell infiltration after 24h of wear persists and amplifies for 24h after lens removal. Corresponding changes in pro-inflammatory cytokine gene expression for IL-17F and RORγt suggest a complex series of events are involved. Further investigation is needed to determine the mechanisms and significance of persistent parainflammatory responses upon lens removal.

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Michel DuPage , Department of Molecular & Cell Biology

Listeria Monocytogenes Persistence Correlates to Tumor Control in Cancer Model

Abstract:

Antigen specific Listeria monocytogenes (Lm) has been used in the past in cancer treatments with limited success. Our approach is different because we use Lm to activate a strong CD8+ T cell response regardless of antigen expression. In collaboration with other lab members, I compared the survival of the Lm strains ΔActA, and ΔActAΔHLY in murine tumors. Previous research from the DuPage lab shows that clearing Lm from tumors has a better effect on tumor control. So to test the ability of the immune system to clear Lm we use several non pathogenic strains of Lm. ΔActA is a non-virulent strain that lacks the polymerization gene and therefore cannot perform cell-to-cell spread. ΔActA ΔHLY also lacks the polymerization and cannot perform cell-to-cell spread. Additionally, it cannot break out of the endosome and ultimately dies in the cell. I cultured MC38 cells and allowed 4 days for them to grow after injecting them into mice. Lm was then injected either intravenously or intratumorally, and at 5 different timepoints, tumors were removed and homogenized. I then plated them for CFUs to indicate if Lm is still present in the tumor after the given number of days. Results have indicated that when injected intratumorally, the ΔActA ΔHLY strain stays at a constant level in the tumors, even after being cleared completely from the periphery. This suggests that some Lm may be existing in the extracellular space of the tumor microenvironment. To investigate this further in future experiments, gentamicin could be injected intravenously after the injection of Lm. This will cause all extracellular bacteria to die and will indicate whether Lm is intracellular or extracellular in the tumors.

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Barbara Baker , Department of Plant and Microbial Biology 

DCL4 is the Hub of a Homeostatic Network that Regulates Plant Defense and RNAi

Abstract:

The prevention of plant disease is of utmost important when considering global food security. Plant resistance to pathogens is governed by their immune systems, part of which is controlled by the R (resistance) – genes encoding for plant innate immune receptors (Nod-Like Proteins and Toll-like Receptors), which recognize pathogen associated molecular patterns (PAMPs) to trigger further immune response. Regulation of these R genes is important to both control pathogen defense and prevent against autoimmunity. In a past study, we studied Dicer-Like Protein 4 (DCL4) and Dicer-Like Protein 2 (DCL2), two antiviral RNA silencing proteins, as they functioned in a novel epigenetic control mechanism of R genes using miRNA mediated gene silencing. In this study, we propose a homeostatic regulatory network involving DCL4/DCL2/RDR6 (RNA Dependent RNA Polymerase 6) which balances antiviral defense and host defense gene expression. 

First, we use a genetic approach to generate genomic resources, using CRIPSR-Cas9 to induce loss of function mutations in each component of the DCL4/DCL2/RDR6 regulatory loop. These edited lines are validated with molecular-genetic approaches and act as a framework for further study. Next, we determine the mechanism of the DCL4/DCL2/RDR6 network, specifically how it controls expression of pathogen defense RNAi genes, R-genes, and other host genes, using comparative genome wide transcriptome analysis. We believe that in healthy environments, RDR6 will inhibit DCL2, but in times of viral infection or DCL4 suppression, DCL2 will be activated, creating 22nt siRNAs which inhibit RDR6, leading to less regulation of R genes, thus enhancing stress response. Finally, we develop new strategies for counteracting pathogen regulation of host target processes by determining the impact of virus infection on virus replication and disease and the impact of viral suppression of the DCL4/DCL2/RDR6 network using comparative analysis of mock and virus infected wild type and mutant plants.

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Michel DuPage , Department of Molecular & Cell Biology

Investigating the Role of EN101 and Hsp90 in Foxp3 Regulation

Abstract:

Regulatory T cells (Tregs) play a key role in modulating immune responses through their suppressive function. While Tregs are essential to prevent autoimmunity, they can also infiltrate tumors and dampen anti-tumor immune responses in cancer. The transcription factor Foxp3 is a master regulator of Treg cell gene expression and modifying its expression levels may be crucial in reprogramming Tregs for cancer therapeutics. EN101 is a cysteine-reactive compound developed by the Nomura Lab and we have demonstrated that it modulates Foxp3 levels. Hsp90 is a chaperone protein involved in protein folding and has been identified as a possible key player in the mechanism of action of EN101. If and how Hsp90 and EN101 are involved in Foxp3 regulation remains unclear. The goal of this project is to investigate the mechanisms involved in the regulation of Foxp3 by EN101 and/or Hsp90.

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John Kuriyan , Department of Molecular and Cell Biology, University of California, Berkeley

Studies on the Chaperoning of BRAF Kinase

Abstract:

BRAF is a protein kinase that is part of the Mitogen Activated Protein Kinase (MAPK) signaling pathway. This pathway is responsible for regulating cell proliferation, differentiation and survival, and gets activated when ligand binds to the receptor thus activating a cascade of proteins, including BRAF, which is activated by a GTP-bound Ras protein. Because of its role in cell proliferation, mutations in the proteins that make up this pathway, resulting in its deregulation, are known drivers of cancer, with the activating mutation of BRAF, V600E, found in approximately 60% of melanomas. Although the structure and function of BRAF is well understood, the mechanisms by which it gets activated remains a mystery. I hypothesize that during its activation, BRAF undergoes an unfolded state where it is chaperoned by the protein HSP90 that poises it for signaling and protects it from degradation. 

Using ratiometric sensors comprising a fluorescently tagged kinase and an untagged control fluorescent protein, I was able to measure the HSP90 dependent stability of different BRAF mutants. Although the literature suggests that BRAF is only a weak client of HSP90, the data from our experiments with the sensor suggest that the kinase domain when not in the auto inhibited state acts as a strong client of HSP90. Thus, BRAF is a context dependent client of this chaperoning apparatus. Further experiments with the activating mutation V600E show that BRAF V600E can synergize with the loss of autoinhibition via the N-terminus to further destabilize the protein, making it more sensitive to HSP90 chaperoning. Therefore, our work suggests that restricting the interaction between BRAF and HSP90 by targeting elements in the N-terminal domain may have therapeutic potential for BRAF-driven cancers.

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Eva Harris , Department of Molecular and Cell Biology, School of Public Health, University of California, Berkeley

Investigating the Role of Lipoproteins in Dengue Virus Nonstructural Protein 1 Activation of the Inflammasome

Abstract:

Dengue is the most prevalent mosquito-borne viral illness globally, with nearly 105 million cases annually. Symptomatic infections with dengue virus (DENV), the causative agent of dengue, typically manifest as an acute febrile illness, but in severe cases can progress to hemorrhage and hypovolemic shock. DENV non-structural protein 1 (NS1) is secreted from infected cells as an oligomer associated with a lipid cargo and has been implicated as a major driver of dengue pathogenesis, activating immune cells and causing pathological endothelial hyperpermeability. Recent work in the Harris lab has shown that DENV NS1 can activate the inflammasome, a class of cytosolic innate immune sensors, inducing the release of the pro-inflammatory cytokine interleukin (IL)-1β. Previous work by others has also demonstrated that DENV NS1 can bind to  lipoproteins in human blood, though how this might affect NS1-induced inflammasome activation is unclear. I now show that high-density lipoproteins (HDL) and low-density lipoproteins (LDL) modulate DENV NS1-induced inflammasome activation in murine bone marrow-derived macrophages. I have found that co-incubation of NS1 and LDL specifically inhibits NS1-induced inflammasome activation while co-incubation of NS1 and HDL does not. Further, LDL appears to regulate the expression of CD14, a lipid binding protein implicated in the NS1-mediated inflammasome activation pathway, on the cell surface, and LDL-intrinsic interactions with macrophages are sufficient to inhibit NS1-induced inflammasome activation. The results of this work provide insight into how the lipoprotein composition of an individual’s blood can affect DENV pathogenesis.