Modulation of human dendritic cell phenotype and function in response to yellow fever vaccination and SARS-CoV-2 infection

Winheim, Elena Patricia (2022): Modulation of human dendritic cell phenotype and function in response to yellow fever vaccination and SARS-CoV-2 infection. Dissertation, LMU München: Medizinische Fakultät

Abstract

Acute viral infections caused by RNA viruses such as flaviviruses (Yellow Fever, Dengue, West Nile) or SARS coronaviruses (SARS-CoV, SARS-CoV-2) represent a major global health threat. The recent worldwide pandemic of SARS-CoV-2 has shown the importance of researching viral infections to identify immune defense mechanisms against these pathogens and understand immune-mediated pathology. In this study, live-attenuated yellow fever 17D (YF17D) vaccination was used as a model of an acute self-limited RNA virus infection in humans, and the role of dendritic cells (DCs) and monocyte subsets in the innate immune response was elucidated using multi-parametric flow cytometry, RNA sequencing, and in vitro experiments. Blood was sampled from vaccinees at time points before (day 0) and after (days 3, 7, 14, 28) YF17D vaccination to analyze the kinetics of the innate immune response. After vaccination with YF17D, DCs and monocytes in the peripheral blood showed concerted activation indicated by the upregulation of CD86 and PD-L1 on the cell surface on day 7 in all DC and monocyte populations (cDC1, cDC2, DC3, tDC, pDC, and classical, intermediate, and non-classical monocytes). At the same time, a robust interferon-induced response was detected in these cells marked by upregulation of Siglec 1 mRNA and surface expression in most cell types - except tDCs and pDCs - and increased expression of a multitude of interferon-induced genes (ISG). A common set of ISGs, consisting of OAS1, OASL, OAS3, RSAD2, IFIT3, IFIT1, and EIF2AK2, was concertedly upregulated in all antigen-presenting subsets analyzed, with peak expression on day 7 after vaccination. Besides this common gene signature induced by vaccination, cell-type-specific effects were also seen, indicating that each DC subset plays a unique role in the innate immune response to YF17D vaccination. Interestingly, DC3, a population with high similarities to both classical monocytes and cDC2, and marked by a CD1c+ CD5- CD14+/- phenotype, showed higher similarity to cDC2 in their transcriptomic response to YF17D vaccination than to classical monocytes, with common gene sets of ISGs and genes relevant for MHC I presentation significantly upregulated on day 7 after vaccination in both cell populations. In vitro experiments using a reporter YF17D virus and flow cytometric detection of YF17D-RNA showed that all DCs and monocytes from the peripheral blood can be infected by YF17D. Infected cells exhibited upregulation of activation markers and secretion of cytokines and chemokines such as type I IFN and CXCL10. Since the viral infection rate was not very high, this direct infection of antigen-presenting cells (APCs) could be used as a mechanism to induce cell activation and allow for antigen presentation. After blocking the IFN-α/β receptor in vitro, high-er YF17D infection rates were found in DCs and monocytes, indicating that the type I interfer-on response is essential in controlling viral replication of YF17D in APCs. Therefore, the strong ISG response found in the transcriptome of peripheral DC and monocyte subsets indicates the induction of an antiviral state in the peripheral blood and suggests highly efficient viral control after vaccination. This posed the question, how viral antigens can be delivered to and efficiently presented by DCs if infection of DCs by YF17D is highly restricted by type I IFNs. It was therefore investigated whether the infection of DCs is more efficient when they are in contact with other infected cells. Indeed, a higher infection rate of DCs and monocytes was achieved after coculture with YF17D-infected cell lines, and this effect depended on cell-to-cell contact. Thus, contact of DCs with YF17D-susceptible cells at the injection site could promote infection and activation of DCs, leading to the presentation of viral antigens to T cells in the draining lymph node. To put the efficient innate immune response to YF17D vaccination into context with another acute RNA virus infection, the innate immune responses to YF17D and SARS-CoV-2 infection were compared. The SARS-CoV-2 viral infection leads to the disease called COVID-19 with different degrees of disease severities and, in some patients, even death. In the early phase after infection, a slow and inefficient control of the virus by the innate immune system may lead to a delayed response which is characterized by hyperinflammation and causes severe immune pathology systemically and in the lung. Compared to the YF17D vaccination, patients with more severe COVID-19 showed low expression of costimulatory molecule CD86 in the cDC2, DC3, and monocytes in the peripheral blood. In contrast, non-hospitalized patients with a mild COVID-19 disease progression showed an upregulation of CD86 similar to what was observed in YF17D vaccinees. The downregulation of CD86 in severe COVID-19 was accompanied by upregulation of PD-L1, which is known to interact with PD-1 on T cells, thereby regulating their inhibiting activation. This altered phenotype of peripheral APCs coincided with a reduced capability of DC3 and monocytes isolated from the blood of COVID-19 patients to stimulate autologous T cell activa-tion and proliferation in vitro, thereby revealing functional impairment of circulating DCs and monocytes in this disease. An increase of Ki67+ cells in both YF17D vaccinees and COVID-19 patients, together with temporary reductions in cDC1 and cDC2 frequencies in the peripheral blood, indicated an increased turnover of the blood DC compartment in acute viral infection. While in YF17D vaccinees only a temporary relative reduction of cDC1 and cDC2 was ob-served, absolute DC numbers and also pDCs were reduced in the peripheral blood of COVID-19 patients. The depletion was transient in patients with a mild disease progression and long-lasting in patients with severe COVID-19. Additionally, a cell population lacking markers of lymphocytes, granulocytes, DCs, and mono-cytes but expressing HLA-DR and Ki67 was also found to be increased in the peripheral blood of COVID-19 patients, but not YF17D vaccinees, as a sign of dysregulated myelopoiesis. Fur-thermore, in COVID-19 patients – but not in YF17D vaccinees – a subset of CD14+ DC3 expanded within the DC3 population and correlated with inflammatory markers and the accumulation of activated Tfh and B cells. Therefore, the innate immune response in COVID-19 patients seems to be a critical factor influencing inflammatory and adaptive immune responses. Dysregulation of innate immune cells as seen by the altered phenotype, impaired function and long-lasting reduction found in DCs and monocytes could lead to an increased susceptibility to sec-ondary infections as a consequence of severe COVID-19. While YF17D vaccination induces a transient coordinated response of blood APC subsets with a peak on day 7 after vaccination, the responses in COVID-19 are long-lasting and show unusual phenotypes of monocytes and DCs accompanied by functional impairment.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: viral infection, dendritic cells, innate immunity, yellow fever, SARS-CoV-2
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 4. Juni 2022
• 1. Bericht­erstatter:in: Krug, Anne