Myasthenia gravis-specific aberrant neuromuscular gene expression by medullary thymic epithelial cells in thymoma

Myasthenia gravis (MG) is a neurological disease caused by autoantibodies against neuromuscular-associated proteins. While MG is frequently developed in thymoma patients, the etiologic factors for MG are not well understood. Here, by constructing a comprehensive atlas of thymoma using bulk and single-cell RNA-seq, we identified ectopic expression of neuromuscular molecules in MG-associated thymoma (MG-thymoma). These molecules were originated from a distinct subpopulation of medullary thymic epithelial cells (mTECs), which we named neuromuscular mTECs (nmTECs). MG-thymoma also exhibited microenvironments dedicated to autoantibody production, including ectopic germinal center formation, T follicular helper cell accumulation, and type 2 conventional dendritic cell migration. Cell-cell interaction analysis also predicted the interaction between nmTECs and T/B cells via CXCL12-CXCR4. The enrichment of nmTECs presenting neuromuscular molecules within MG-thymoma was further confirmed by immunohistochemically and by cellular composition estimation from MG-thymoma transcriptome. Altogether, this study suggests that nmTECs play a significant role in MG pathogenesis via ectopic expression of neuromuscular molecules.

A controversial role of neutrophils in tuberculosis infection pathogenesis

Tuberculosis (TB) continues to be an important and unresolved medical problem. About a quarter of mankind is infected with Mycobacterium tuberculosis, and about 5–10% of these people eventually develop TB. Macrophages and CD4+ T cells are considered the key cells providing defense against TB infection. The role of neutrophils in TB is less well defined. Neutrophils are short-lived granulocytes among first migrate into the infectious lung tissue and phagocy tose mycobacteria. On the one hand, there is evidence for protective role of neutrophils in TB released via anti-microbial peptides inhibiting mycobacterial growth, up-regulation of CD4+ T-cell activation, and dendritic cell migration in the lymph nodes. On the other hand, infection of genetically TB susceptible animals leads to an overwhelming lung neutrophil inflammation, development of necrotic granulomata, and a rapid death. Neutrophils act directly or indirectly on mycobacteria by different oxidative or other reactions including neutrophil extracellular traps (NETs) formation. Phagocytosis of mycobacteria by neutrophils is accompanied by the production of pro-inflammatory factors, thus making neutrophils active participants of inflammation in all stages of the infectious process. Finally, neutrophils die by apoptosis or necrosis. Necrosis of neutrophils, which is activated by reactive oxygen species, also prolongs the inflammation. In this way, there is strong evidence that neutrophils are the cells involved in the transition of infection to the terminal stage, participating in lung tissue destruction. Although neutrophils evolutionary developed many ways to resist pathogens, it is likely, that neutrophils do not possess sufficient anti-mycobactericidal capacities due to the development of many adaptations allowing mycobacteria to survive inside the neutrophils. Neutrophils effectively phagocytose but poorly kill mycobacteria, thus hiding bacilli from more efficient killers, macrophages, and playing the role of the “Trojan Horse”. In this review, we summarize the data on the involvement of neutrophils in TB inflammation. We discuss their ambiguous role in pathogenesis which depends upon mycobacterial virulence, host genetics, dynamics of migration to inflammatory foci, and persistence during initial and chronic stages of the infectious process.

EXTH-44. SYSTEMIC CCL3 TREATMENT ENHANCES IMMUNOTHERAPY EFFICACY THROUGH IMPROVED DENDRITIC CELL MIGRATION

INTRODUCTION Dendritic cell (DC) vaccines have shown marginal success in treating glioblastoma (GBM), with inefficient vaccine migration a major limitation. Prior evidence from our clinical trials demonstrated that tetanus diphtheria (Td) preconditioning produced greater DC migration to vaccine draining lymph nodes (VDLNs) and long-term survival. Greater DC numbers reaching VDLNs was also associated with long-term survival. We found from preclinical studies and our patients that increased DC migration was dependent upon the chemokine (C-C motif) ligand 3 (CCL3). METHODS The effect of systemic CCL3 treatment on DC vaccine migration (n=5), antigen-specific T cell responses (n=5) and efficacy against orthotopic GL261-OVA and SMA560 tumors (n=10) was studied in C57Bl/6 and VMdK mice. DCs were electroporated with OVA-mRNA or pulsed with ODC1 neoantigen peptide. Median overall survival (mOS) was measure in days (d) post-intracranial implantation. RESULTS Intravenous CCL3 at the time of intradermal DC vaccination resulted in a dose-dependent increase in migration to VDLN (10ug p=0.036, 20ug p< 0.0001, 50ug p< 0.0001). Mean migration levels following CCL3 treatment were similar to Td-preconditioning (p=0.52) but showed significantly less variability between mice. Combined CCL3 and DC vaccination generated more tumor antigen-specific CD8+IFNγ+ T cells 7 days compared to DC vaccine alone (p=0.0045). CCL3+OVA-DC treatment resulted in significantly greater survival compared to OVA-DC alone (mOS 37 vs 19.5 d; p=0.0174) in established GL261-OVA. CCL3 treatment increased survival in mice with established SMA560 tumors treated with neoantigen ODC1 peptide-pulsed DCs (Tumor alone mOS: 21d, DCvac: 25d, CCL3+DCvac: 48d, p=0.002). CONCLUSIONS These data combined with previous success of our DC vaccine clinical trials reflect the potency of CCL3 to enhance DC vaccine-specific migration, immune responses and survival. CCL3 is a novel and safe adjuvant to overcome prior limitations in DC vaccine therapy and may be translatable to increase heterogeneous tumor antigen presentation following vaccine-targeted tumor killing.

CAL-1 as Cellular Model System to Study CCR7-Guided Human Dendritic Cell Migration

Dendritic cells (DCs) are potent and versatile professional antigen-presenting cells and central for the induction of adaptive immunity. The ability to migrate and transport peripherally acquired antigens to draining lymph nodes for subsequent cognate T cell priming is a key feature of DCs. Consequently, DC-based immunotherapies are used to elicit tumor-antigen specific T cell responses in cancer patients. Understanding chemokine-guided DC migration is critical to explore DCs as cellular vaccines for immunotherapeutic approaches. Currently, research is hampered by the lack of appropriate human cellular model systems to effectively study spatio-temporal signaling and CCR7-driven migration of human DCs. Here, we report that the previously established human neoplastic cell line CAL-1 expresses the human DC surface antigens CD11c and HLA-DR together with co-stimulatory molecules. Importantly, if exposed for three days to GM-CSF, CAL-1 cells induce the endogenous expression of the chemokine receptor CCR7 upon encountering the clinically approved TLR7/8 agonist Resiquimod R848 and readily migrate along chemokine gradients. Further, we demonstrate that CAL-1 cells can be genetically modified to express fluorescent (GFP)-tagged reporter proteins to study and visualize signaling or can be gene-edited using CRISPR/Cas9. Hence, we herein present the human CAL-1 cell line as versatile and valuable cellular model system to effectively study human DC migration and signaling.

First person – Katharina Vestre

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Katharina Vestre is first author on ‘ Rab7b regulates dendritic cell migration by linking lysosomes to the actomyosin cytoskeleton’, published in JCS. Katharina is a PhD student in the lab of Cinzia Progida at the Department of Biosciences, University of Oslo, Norway, investigating the coordination between intracellular traffic and the cytoskeleton, and how this affects processes such as cell division and migration.

Rab7b regulates dendritic cell migration by linking lysosomes to the actomyosin cytoskeleton

Lysosomal signaling facilitates the migration of immune cells by releasing calcium to activate the actin-based motor myosin II at the cell rear. However, how the actomyosin cytoskeleton physically associates to lysosomes is unknown. We have previously identified myosin II as a direct interactor of Rab7b, a small GTPase that mediates the transport from late endosomes/lysosomes to the TGN. Here, we show that Rab7b regulates the migration of dendritic cells (DCs) in 1- and 3-dimensional environments. DCs are immune sentinels that transport antigens from peripheral tissues to lymph nodes to activate T lymphocytes and initiate adaptive immune responses. We found that lack of Rab7b reduces myosin II light chain phosphorylation and the activation of the transcription factor EB (TFEB), which controls lysosomal signaling and is required for fast DC migration. Furthermore, we demonstrate that Rab7b interacts with the lysosomal calcium channel TRPML1, enabling the local activation of myosin II at the cell rear. Altogether, our findings identify Rab7b as the missing physical link between lysosomes and the actomyosin cytoskeleton, allowing control of immune cell migration through lysosomal signaling.

Leishmania-Induced Dendritic Cell Migration and Its Potential Contribution to Parasite Dissemination

Leishmania, an intracellular parasite species, causes lesions on the skin and in the mucosa and internal organs. The dissemination of infected host cells containing Leishmania is crucial to parasite survival and the establishment of infection. Migratory phenomena and the mechanisms underlying the dissemination of Leishmania-infected human dendritic cells (hDCs) remain poorly understood. The present study aimed to investigate differences among factors involved in hDC migration by comparing infection with visceral leishmaniasis (VL) induced by Leishmaniainfantum with diverse clinical forms of tegumentary leishmaniasis (TL) induced by Leishmaniabraziliensis or Leishmania amazonensis. Following the infection of hDCs by isolates obtained from patients with different clinical forms of Leishmania, the formation of adhesion complexes, actin polymerization, and CCR7 expression were evaluated. We observed increased hDC migration following infection with isolates of L. infantum (VL), as well as disseminated (DL) and diffuse (DCL) forms of cutaneous leishmaniasis (CL) caused by L. braziliensis and L. amazonensis, respectively. Increased expression of proteins involved in adhesion complex formation and actin polymerization, as well as higher CCR7 expression, were seen in hDCs infected with L. infantum, DL and DCL isolates. Together, our results suggest that hDCs play an important role in the dissemination of Leishmania parasites in the vertebrate host.