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Understanding of the factors governing immune responses in cancer remains incomplete, limiting patient benefit. In this study, we used mass cytometry to define the systemic immune landscape in response to tumor development across five tissues in eight mouse tumor models. Systemic immunity was dramatically altered across models and time, with consistent findings in the peripheral blood of patients with breast cancer. Changes in peripheral tissues differed from those in the tumor microenvironment. Mice with tumor-experienced immune systems mounted dampened responses to orthogonal challenges, including reduced T cell activation during viral or bacterial infection. Antigen-presenting cells (APCs) mounted weaker responses in this context, whereas promoting APC activation rescued T cell activity. Systemic immune changes were reversed with surgical tumor resection, and many were prevented by interleukin-1 or granulocyte colony-stimulating factor blockade, revealing remarkable plasticity in the systemic immune state. These results demonstrate that tumor development dynamically reshapes the composition and function of the immune macroenvironment. Primary tumor presence and progression shape the systemic immune landscape and immune responses to pathogens in multiple murine tumor models.

Silencing remains a significant challenge for exogenous gene expression, limiting both the penetrance and expressivity of transgenes. In particular, silencing of Cas9 expression is a major technical limitation for many gene editing and CRISPR screening applications. Here, we demonstrate that including introns in Cas9 expression cassettes significantly reduces silencing across multiple cell lines. Notably, the incorporation of an intron into a CRISPRa construct results in reduced silencing, increased expression levels, and markedly enhanced activation of target genes. We investigate diverse intron sequences and discover that T-rich introns over 2 kb confer the greatest protection against silencing. In addition, we find that introns can work synergistically with chromatin opening elements to further mitigate silencing, suggesting regulatory mechanisms are acting at both the DNA and RNA level to silence exogenous genes. Our work highlights the potential of introns to optimize genetic constructs for enhanced expression and improved cellular engineering requiring constitutive expression of large transgenes. Silencing of transgenes such as Cas9 limits gene editing and CRISPRa applications. Here, the authors show that adding intronic sequences reduces silencing and boosts transgene expression, enabling improved CRISPRa-mediated gene activation and more stable expression of the transgene over time.

The precise strategies that intracellular pathogens use to exit host cells have a direct impact on their ability to disseminate within a host, transmit to new hosts, and engage or avoid immune responses. The obligate intracellular bacterium Chlamydia trachomatis exits the host cell by two distinct exit strategies, lysis and extrusion. The defining characteristics of extrusions, and advantages gained by Chlamydia within this unique double‐membrane structure, are not well understood. Here, we define extrusions as being largely devoid of host organelles, comprised mostly of Chlamydia elementary bodies, and containing phosphatidylserine on the outer surface of the extrusion membrane. Extrusions also served as transient, intracellular‐like niches for enhanced Chlamydia survival outside the host cell. In addition to enhanced extracellular survival, we report the key discovery that chlamydial extrusions are phagocytosed by primary bone marrow‐derived macrophages, after which they provide a protective microenvironment for Chlamydia. Extrusion‐derived Chlamydia staved off macrophage‐based killing and culminated in the release of infectious elementary bodies from the macrophage. Based on these findings, we propose a model in which C. trachomatis extrusions serve as “trojan horses” for bacteria, by exploiting macrophages as vehicles for dissemination, immune evasion, and potentially transmission. Chlamydia possess two conserved mechanisms for exiting host cells, one of which (extrusion) releases bacteria into a membrane‐bound compartment. Here we demonstrate that extrusions provide a microenvironment that sustains infectivity of extracellular Chlamydia during cell to cell traversal, and that extrusions present signals for phagocytosis by macrophages. Importantly, macrophages are unable to completely kill these internalized bacteria, thus raising potentially novel strategies for Chlamydia dissemination within a host.

Pancreatic ductal adenocarcinoma (PDAC), a malignancy refractory to most therapies including immune checkpoint blockade (ICB) therapy, utilizes diverse mechanisms to evade immune clearance. One mechanism involves reduced presentation of tumor specific antigens by Major Histocompatibility Complex Class I (MHC-I) to immune cells. Many cancers alter MHC-I expression via genetic or epigenetic silencing, however changes in MHC-I trafficking can also profoundly influence antigen presentation at the cell surface and is a previously underappreciated mechanism of MHC-I regulation in cancer.My dissertation uncovers a role for enhanced autophagy/lysosome function in immune evasion through selective targeting of MHC-I molecules for degradation. Prior studies have shown that highly aggressive PDAC cells and tumors upregulate autophagy, an evolutionarily conserved self-recycling pathway that is hijacked by cancer cells to sustain metabolic fitness. In addition to the metabolic benefits tumor cells receive, autophagy and lysosomal activity have utilized these processes to gain a growth advantage by facilitating degradation and recycling of diverse intracellular materials. My data demonstrates that MHC-I molecules are selectively targeted for lysosomal degradation through an autophagy-dependent mechanism that involves the autophagy cargo receptor NBR1. PDAC cells display reduced MHC-I cell surface expression and instead demonstrate predominant localization within autophagosomes and lysosomes. Notably, autophagy inhibition restores cell surface MHC-I expression, enhances anti-tumor CD8+ T cell responses in vitro and in vivo, and importantly, sensitizes PDAC tumors to dual immune checkpoint blockade (ICB). Our data on immune evasion adds to the growing list of cell- autonomous functions of the autophagy/lysosome system in supporting PDAC tumorigenesis.To identify the distinct molecular mechanisms of MHC-I regulation in PDAC, we combined a whole-genome CRISPRi screen and Turbo-ID proximity-dependent proteomics to determine regulators and interactors of MHC-I, respectively. From these two datasets, 101 overlapping candidates were identified, many of which were related to post-translational modification (PTM), trafficking machinery, and kinase regulation. The gene candidates likely control MHC-I by diverting plasma membrane localization to degradative organelles. Several gene candidates associated with E3 ubiquitin ligase and kinase signaling were validated and show increased plasma membrane when knocked down or pharmacologically inhibited. Using these two datasets as a resource of MHC-I regulation can help identify PDAC-specific mechanisms that facilitate altered trafficking of MHC-I.Findings from this study will lead to a better understanding of PDA pathophysiology and have the potential to inform the development of rational combination therapies that restore MHC-I cell surface localization, thereby rendering PDA cells more susceptible to immunotherapy. In particular, results from this research project will help to determine the causes of aberrant intracellular localization of MHC-I and can help explain MHC-I is unable to traffic normally. These studies also lay the foundation for mechanisms of immune evasion in other aggressive cancers.

Immune evasion is a major obstacle for cancer treatment. Common mechanisms of evasion include impaired antigen presentation caused by mutations or loss of heterozygosity of the major histocompatibility complex class I (MHC-I), which has been implicated in resistance to immune checkpoint blockade (ICB) therapy 1 – 3 . However, in pancreatic ductal adenocarcinoma (PDAC), which is resistant to most therapies including ICB 4 , mutations that cause loss of MHC-I are rarely found 5 despite the frequent downregulation of MHC-I expression 6 – 8 . Here we show that, in PDAC, MHC-I molecules are selectively targeted for lysosomal degradation by an autophagy-dependent mechanism that involves the autophagy cargo receptor NBR1. PDAC cells display reduced expression of MHC-I at the cell surface and instead demonstrate predominant localization within autophagosomes and lysosomes. Notably, inhibition of autophagy restores surface levels of MHC-I and leads to improved antigen presentation, enhanced anti-tumour T cell responses and reduced tumour growth in syngeneic host mice. Accordingly, the anti-tumour effects of autophagy inhibition are reversed by depleting CD8 + T cells or reducing surface expression of MHC-I. Inhibition of autophagy, either genetically or pharmacologically with chloroquine, synergizes with dual ICB therapy (anti-PD1 and anti-CTLA4 antibodies), and leads to an enhanced anti-tumour immune response. Our findings demonstrate a role for enhanced autophagy or lysosome function in immune evasion by selective targeting of MHC-I molecules for degradation, and provide a rationale for the combination of autophagy inhibition and dual ICB therapy as a therapeutic strategy against PDAC. Inhibition of the autophagy–lysosome system upregulates surface expression of MHC class I proteins and enhances antigen presentation, and evokes a potent anti-tumour immune response that is mediated by CD8 + T cells.

Pancreatic ductal adenocarcinoma (PDA) evades immune detection partly via autophagic capture and lysosomal degradation of major histocompatibility complex class I (MHC-I). Why MHC-I is susceptible to capture via autophagy remains unclear. By synchronizing exit of proteins from the endoplasmic reticulum (ER), we show that PDAC cells display prolonged retention of MHC-I in the ER and fail to efficiently route it to the plasma membrane. A capture-complex composed of NBR1 and the ER-phagy receptor TEX264 facilitates targeting of MHC-I for autophagic degradation, and suppression of either receptor is sufficient to increase total levels and re-route MHC-I to the plasma membrane. Binding of MHC-I to the capture complex is linked to antigen presentation efficiency, as inhibiting antigen loading via knockdown of TAP1 or beta 2-Microglobulin led to increased binding between MHC-I and the TEX264-NBR1 capture complex. Conversely, expression of ER directed high affinity antigenic peptides led to increased MHC-I at the cell surface and reduced lysosomal degradation. A genome-wide CRISPRi screen identified NFXL1, as an ER-resident E3 ligase that binds to MHC-I and mediates its autophagic capture. High levels of NFXL1 are negatively correlated with MHC-I protein expression and predicts poor patient prognosis. These data highlight an ER resident capture complex tasked with sequestration and degradation of non-conformational MHC-I in PDAC cells, and targeting this complex has the potential to increase PDAC immunogenicity.

Microglia are the immune cells of the central nervous system and are thought to be key players in both physiological and disease conditions. Several microglial features are poorly conserved between mice and human, such as the function of the neurodegeneration-associated immune receptor Trem2. Induced pluripotent stem cell (iPSC)-derived microglia offer a powerful opportunity to generate and study human microglia. However, human iPSC-derived microglia often exhibit activated phenotypes , and assessing their impact on other brain cell types remains challenging due to limitations in current co-culture systems. Here, we developed fully defined brain microtissues, composed of human iPSC-derived neurons, astrocytes, and microglia, co-cultured in 2D or 3D formats. Our microtissues are stable and self-sufficient over time, requiring no exogenous cytokines or growth factors. All three cell types exhibit morphologies characteristic of their environment and show functional properties. Co-cultured microglia develop more homeostatic phenotypes compared to microglia exposed to exogenous cytokines. Hence, these tri-cultures provide a unique approach to investigate cell-cell interactions between brain cell types. We found that astrocytes and not neurons are sufficient for microglial survival and maturation, and that astrocyte-derived M-CSF is essential for microglial survival. Single-cell and single-nucleus RNA sequencing analyses nominated a network of reciprocal communication between cell types. Brain microtissues faithfully recapitulated pathogenic α-synuclein seeding and aggregation, suggesting their usefulness as human cell models to study not only normal but also pathological cell biological processes.

Harnessing immune defense mechanisms has revolutionized cancer therapy, but our understanding of the factors governing immune responses in cancer remains incomplete, limiting patient benefit. Here, we use mass cytometry to define the organism-wide immune landscape in response to tumor development across five tissues in eight tumor models. Systemic immunity was dramatically altered across mouse models and cancer patients, with changes in peripheral tissues differing from those in the tumor microenvironment and taking place in phases during tumor growth. This tumor-experienced immune system mounted dampened responses to orthogonal challenges, including reduced T cell activation during viral or bacterial infection. Disruptions in T cell responses were not cell-intrinsic but rather due to reduced responses in antigen-presenting cells (APCs). Promoting APC activation was sufficient to restore T cell responses to orthogonal infection. All systemic immune changes were reversed with surgical tumor resection, revealing remarkable plasticity in the systemic immune state, which contrasts with terminal immune dysfunction in the tumor microenvironment. These results demonstrate that tumor development dynamically reshapes the composition and function of the immune macroenvironment.

All obligate intracellular pathogens must exit their host cells in order to propagate and survive as a species; the precise strategies they use have a direct impact on their ability to disseminate within a host, transmit to new hosts, and engage or avoid immune responses. The obligate intracellular bacterium Chlamydia trachomatis exits the host cell by two distinct exit strategies, lysis and extrusion. Despite being equally active pathways, lysis and extrusion differ greatly in their mechanisms. The defining characteristics of extrusions, and advantages gained by Chlamydia within this unique double-membrane structure are not well understood. Here, we present data that defines extrusions as being largely devoid of host organelles, comprised mostly of Chlamydia elementary bodies, and containing phosphatidylserine on the outer surface of the extrusion membrane. Towards defining a functional role for extrusions in Chlamydia pathogenesis, we demonstrate that extrusions confer significant infectious advantages for Chlamydia by serving as transient, intracellular-like niches for extracellular Chlamydia, as compared to Chlamydia that would otherwise exit by lysing the host cell. In addition to enhanced survival outside of the host cell, we report the key discovery that chlamydial extrusions can be engulfed by primary bone marrow-derived macrophages, after which they provide a protective microenvironment for Chlamydia. Extrusion-derived Chlamydia were able to stave off macrophage based killing beyond 8 h, and culminated in the release of infectious EB from the macrophage. Based on these findings, we propose a model in which a major outcome of Chlamydia exiting epithelial cells inside extrusions is to hijack macrophages as vehicles for dissemination within the host.