Immune suppression during chronic lymphocytic choriomeningitis virus infection and response to unrelated ectromelia virus infection

Universidade do Minho Escola de Medicina Pedro Filipe Alves Peixoto Immune Suppression During Chronic Lymphocytic Choriomeningitis Virus Infection and Response to Unrelated Ectromelia Virus Infection Julho de 2020 UMinho |2020 Pedro Peixoto Immune Suppresion During Chronic Lymphocytic Choriomeningitis Virus Infection and Response to Unrelated Ectromelia Virus Infection Universidade do Minho Escola de Medicina Pedro Filipe Alves Peixoto Immune Suppression During Chronic Lymphocytic Choriomeningitis Virus Infection and Response to Unrelated Ectromelia Virus Infection Tese de Doutoramento em Medicina Trabalho efetuado sob a orientação de Professor Doutor Luis J. Sigal Professora Doutora Margarida Correia-Neves Julho de 2020 ii DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Este é um trabalho académico que pode ser utilizado por terceiros desde que respeitadas as regras e boas práticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licença abaixo indicada. Caso o utilizador necessite de permissão para poder fazer um uso do trabalho em condições não previstas no licenciamento indicado, deverá contactar o autor, através do RepositóriUM da Universidade do Minho. Licença concedida aos utilizadores deste trabalho Atribuição-NãoComercial-CompartilhaIgual CC BY-NC-SA https://creativecommons.org/licenses/by-nc-sa/4.0/ [Esta licença permite que outros remisturem, adaptem e criem a partir do seu trabalho para fins não comerciais, desde que lhe atribuam a si o devido crédito e que licenciem as novas criações ao abrigo de termos idênticos.] iii Acknowledgments I want to acknowledge Professor Luis J. Sigal for your strenuous mentorship and continuous guidance. You are my role model as a scientist. Thank you for devoting so much patience, hard work and time to improve me. Thank you, Professor Margarida Correia-Neves, for believing in my capabilities and pushing forward for me to succeed as a scientist. If it were not for you, I would have never started this endeavor. To all the people who I worked with at Professor Sigal’s laboratory. I want to acknowledge Cory Knudson, Maria Férez, Carolina Melo-Silva, Eric B Wong, Colby Stotesbury, Brian Montoya, Lingjuan Tang and Ni Meng. I believe we worked as a fine-tuned machine in which all took their work seriously, while building amazing memories. Thank you to my thesis committee, Professor Kerry Campbell, Professor Cristopher Snyder, Professor Gudrun Debes and Professor Kishore Alugupalli, for your critical insight and helpful questions. To the MD/PhD program. I leave a special note of acknowledgment to Professor Nuno Sousa and Professor Tiago Gil, as well as to the School of Medicine at University of Minho (EM-UM), for your continuous efforts to push forward for improved academic excellence of your students. Thank you to Professor Gerald B. Grunwald, for continuously supporting the EM-UM MD/PhD program and students at Thomas Jefferson University. To my colleagues of the MD/PhD program, for your enthusiasm, companionship, and support throughout these last few years, particularly to Emanuel Novais, Tiago Brás, Sofia Santos, Sofia Dantas, Tiago Mota, Daniel Machado and Jorge Silva. Finally, to my family. Thank you for being my role model in life and the safe harbor I can always trust in. Thank you for always pushing forward for me to be a better person, for your love that drives me in all aspects of my life. I hope this is just another step in a life plentiful of shared memories and love. Financial support for my PhD was provided by Fundação para a Ciência e Tecnologia (FCT) with the fellowship PD/BD/128078/2016, awarded through the MD/PhD program of the EM-UM. iv STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. v Supressão imunológica durante a infeção crónica com o vírus da coriomeningite linfocítica e resposta à infeção não relacionada pelo vírus ectromelia Infeções virais persistentes, incluindo as causadas por vírus pouco patogénicos como o citomegalovírus, mas também outros mais virulentos para o hospedeiro, como o vírus da hepatite B (VHB) ou da hepatite C (VHC) podem resultar em disfunção imunitária e aumentar a suscetibilidade a infeções não relacionadas ou a cancro. No trabalho aqui apresentado utilizámos o modelo de infeção crónica em murganhos pelo vírus da coriomeningite linfocítica (LCMV) clone 13 (CL13) para estudar a resposta à co-infeção pelo vírus ectromelia (ECTV), o agente da mousepox . Os nossos resultados mostram que murganhos cronicamente infetados com CL13 são mais suscetíveis à infeção por ECTV, do que animais previamente saudáveis ou recuperados de infeção pela estirpe Armstrong (Arm) do LCMV. A infeção crónica por CL13 traduz-se numa redução acentuada dos números e frequências de células Natural Killer (NK), que apresentam um fenótipo marcadamente imaturo e que são incapazes de responder à infeção subsequente por ECTV, com níveis reduzidos de maturação, ativação e citotoxicidade, com produção reduzida de granzima (Gzm) B e interferão (IFN) γ, em contraste com as células NK de murganhos saudáveis ou recuperados após infeção por Arm. Murganhos infetados cronicamente com CL13 apresentam células T CD8+ que respondem insuficientemente a uma infeção secundária por ECTV, com níveis de ativação fracos, frequências reduzidas e alterações fenotípicas do compartimento de células CD44+GzmB+, bem como com reduzida expansão de células TCR-epítopo especificas. No entanto, é possível induzir uma resposta T CD8+ TCRepítopo especifica forte aquando da infeção secundária de murganhos infetados cronicamente com CL13 com um vírus atenuado ECTV36, conferindo resistência à infeção posterior com ECTV. O nosso trabalho sugere que a suscetibilidade a infeções oportunistas em indivíduos previamente persistentemente infetados pode estar associada a defeitos das células NK e T CD8+. Portanto, será possível que doentes imunodeprimidos por estas razões possam ser protegidos de infeções oportunistas desde que vacinas efetivas sejam utilizadas. Palavras chave: Infeção viral crónica; Infeção viral aguda; LCMV; ECTV; células NK, células T vi Immune suppression during chronic lymphocytic choriomeningitis virus infection and response to unrelated ectromelia virus infection Persistent viral infections, from the relatively nonpathogenic cytomegalovirus, to others that severely impact the host, including the human immunodeficiency virus (HIV), even under antiretroviral therapy (ART), hepatitis B virus (HBV) or hepatitis C virus (HCV), can result in immune dysfunction that predisposes to severe infections with unrelated pathogens and to cancer. Here we use the mouse model of infection with the Clone 13 (CL13) strain of lymphocytic choriomeningitis virus (LCMV) to study how the immune system of the persistently infected wild-type (WT) mouse host responds to unrelated ectromelia virus (ECTV) infection, the agent of mousepox in the mouse. We show that WT mice chronically infected with CL13 are fully susceptible to ECTV infection and succumb to mousepox, contrasting with previously naïve mice or convalescent from acute LCMV infection with the Armstrong strain (Arm), due to a multitude of defects in their immune. We show that CL13 infection leads to a severe reduction of the numbers and frequencies of Natural Killer (NK) cells, that bear a highly immature phenotype and fail to respond to secondary ECTV infection, with impaired maturation and activation, and decreased cytotoxic function, due to reduced production of granzyme B and interferon (IFN) γ, in contrast with NK cells in Arm-convalescent or previously naïve mice. Moreover, in CL13 chronically infected mice, CD8+ T cells respond poorly to secondary infection with the ECTV, with impaired activation, reduced frequencies and phenotypic changes of highly functional CD44+ GzmB+ cells and reduced clonal expansion of TCR epitope specific CD8+ T cells. We show that it is possible to induce a potent ECTV-specific CD8+ T cell response if CL13 infected mice are challenged with a highly attenuated ECTV36 virus strain, resulting in full resistance to subsequent ECTV infection. Our work suggests that susceptibility to opportunistic infections in the persistently infected individuals may be linked to NK cell and CD8+ T cell dysfunction. Moreover, it is possible that immunocompromised persistently infected individuals may be protected from opportunistic infections provided effective vaccines are used. Keywords: Chronic Viral Infection; Acute Viral Infection; LCMV; ECTV; NK cells; T cells. vii Abbreviations AIDS – Acquired Immune Deficiency Syndrome APC – Antigen Presenting Cell Arm – Armstrong strain BrdU – Bromodeoxyuridine BTLA – B and T Lymphocyte Attenuator CCL – Chemokine (C-C motif) Ligand CCL – Chemokine Ligand CCR – C-C Chemokine Receptor CL13 – clone 13 CMV – Cytomegalovirus CTL – Cytotoxic T Lymphocytes CTLA-4 – Cytotoxic T-lymphocyte-associated protein 4 CXCL – Chemokine (C-X-C motif) Ligand CXCR – C-X-C Chemokine Receptor DCs – Dendritic Cells DG – dystroglycan dLN – draining Lymph Node dpi – days post infection E – Glutamate EBV – Epstein-Barr Virus ECTV – Ectromelia Virus EOMES – Eomesodermin F – Phenylalanine GP – Glycoprotein Gzm – Granzyme HAART – Highly Active Antiretroviral Therapies HBV – Hepatitis B Virus HCMV – Human Cytomegalovirus HCV – Hepatitis C Virus HHV – Human Herpesvirus HIF – Hypoxia-Inducible Factors HIV – Human Immunodeficiency Virus HSV – Herpes Simplex Virus IAV – Influenza A virus IFN – Interferon IFNAR – Interferon-α/β Receptor Ig – Immunoglobulin IL – interleukin IL-10R – IL-10 Receptor iMOs – inflammatory Monocytes ISG – interferon Stimulated Gene K – Lysine KLRG1 – Killer cell Lectin-like Receptor subfamily G member 1 L – Leucine LAG – Lymphocyte Activation Gene protein LCMV - Lymphocytic Choriomeningitis Virus MCMV – Mouse Cytomegalovirus MHC – Major Histocompatibility Complex NK cells – Natural Killer cells NP – Nucleoprotein OPV – orthopoxvirus PD1 – Programmed cell Death protein 1 viii pDCs – plasmocytoid DCs PD-L1 – Programmed Death-Ligand 1 PFU – plaque forming units Prf - Perforin PVPichinde virus SIRP – Signal Regulatory Protein α TCF – Transcription factor T cell factor TCR – T cell receptor TFTranscription factor TGF-β – Tumour Growth Factor β TIGIT - T cell immunoreceptor with Ig and ITIM domains TIM-3 – T-cell limmunoglobulin and mucindomain containing protein 3 TLR – Toll-Like Receptor TNF – Tumour Necrosis Factor VHL – Von Hippel–Lindau Tumor Supressor VSV– Vesicular Stomatitis Virus VZV – Varicella Zoster Virus WHO – World Health Organization WT – Wild Type 15 Part I – Introduction 16 CHAPTER I. Chronic viral infections: epidemiology and overview It is estimated that every person in the world carries 8-12 long-lasting infections, at any given time. Currently thousands of millions of people are infected with a chronic virus, regardless of their pathogenicity to the host (Figure 1). Some of these chronic viral infections are associated with obvious increased morbidity and mortality and received strong attention like the human immunodeficiency virus (HIV) among a few others. Yet, most of the chronic viral infections cause a less severe and obvious effect and consequently receive relatively low attention. However, all these chronic infections impact the host immune system and need to be carefully addressed. Figure 1. The estimated global prevalence of chronic infections. Estimates consider a world population of 6.75 billion people. Extracted from (1). 17 Human immunodeficiency virus (HIV) Since the 1980s, when HIV was described and the first acute immunodeficiency syndrome (AIDS) victims perished to the destructive effects of HIV, much has changed in the treatments used. The emergence of highly active antiretroviral therapies (HAART) in the 1990s transformed HIV infection from an eminently lethal disease against which little could be done, to a chronic infection that can be controlled, with patients having a lifespan very close to the population average, despite premature aging, drug treatment toxicity, decreased quality of life and increased frequency of comorbidities, including increased susceptibility to opportunistic infections and cancer (Table 1 and (2)). In 2018, there were 37.9 million people living with HIV, and of those, 1.7 million were newly infected cases. Therefore, the number of people infected with HIV is still increasing, with a 20% increase in the period between 2010 and 2018. Despite current treatment therapies, 0.8 million people perished due to HIV-related diseases, a problem that is aggravated by the fact many people have limited access to treatment or have it in an intermittent manner. However, this still represents a 33% decrease in mortality from 2010 (2). Infection with HIV leads to severe immunosuppression and ultimately death of the host, if left without treatment. HIV infects cells by binding to the CD4 receptor in CD4+ T cells, with the help of the CCR5 and CXCR4 co-receptors. After an initial period of relative viral control, the numbers of CD4+ T cells progressively plummet (3). Moreover, the remaining CD4+ T cells have lower potential to respond to new stimuli and produce a polyfunctional response (-4). HIV also induces severe dysfunction of CD8+ T cells, B cells and natural killer cells (NK cells) (3). All of this results in severe immunosuppression (3) and contributes to the increased susceptibility to opportunistic infections, cancer and comorbidities, including metabolic diseases, higher levels of inflammation and hyperstimulation of the immune system, together with higher frailty levels at later timepoints in life (5–7). Unfortunately, despite the effectiveness of current HAART in recovering CD4+ T cell numbers in most patients, the higher risks of development of disease in HIV infected individuals is not fully eliminated (5). 18 Table 1. Quality of life in HIV infected individuals. Extracted from (5). 19 Viral hepatitis Chronic viral hepatitis virus infections are a major burden worldwide, despite an effective vaccine for Hepatitis B (HBV), and curative treatment for Hepatitis C (HCV). In 2015, the World Health Organization (WHO) estimated that 257 million people were living with HBV and 71 million with HCV infection. Together the two infections were responsible for 1.34 million fatalities. To this scenario, new cases are added every year. In 2015, the WHO estimated 1.75 million people were newly diagnosed with HCV infection (8). Figure 2. Mechanisms of suppression of T cells in chronic HBV infection. Extracted from (9). Hepatitis viruses can induce severe immune suppression (Figure 2 and (10)). For instance HBV chronic infection leads to impairment of several immune cells, including monocytes, macrophages, dendritic cells (DCs), NK cells and T cells (10). HBV drives DCs to produce tumor growth factor β (TGF-β) and interleukin (IL) 10 (11,12), potent immune suppressors, which contributes to exhaustion of CD4+ T cells and CD8+ T cells (9,11) and weakened function of NK cells (13). NK cells show reduced production of Interferon (IFN) γ and increased IL10 production (10). In infection with either HBV or HCV, CD8+ T cells increased expression of several suppressing receptors, including programmed cell death protein 1 (PD1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), 2B4 and 20 T-cell immunoglobulin and mucin-domain containing protein 3 (TIM-3) (14,15). In hepatitis, CD8+ T cells have reduced production of IFN-γ, tumour necrosis factor (TNF) α and IL21, and reduced proliferative capability (9). There are 2.7 million people co-infected with HIV and HBV and 2.3 million with HIV and HCV (8). These co-infections are associated with worse prognosis of HIV infection progression (16). Among those coinfected with both, HIV and either HBV or HCV, the prevalence of AIDS complications, including several bacterial infections, mycosis and toxoplasmosis, is higher than those only infected with HIV (16). Chronic HCV infection is reported to impact the immune response to vaccination against unrelated pathogens. For example, it severely decreases immunoglobulin (Ig) G seroconversion following anti-Hepatitis A and anti-Influenza vaccination and also reduces the duration of protection (17–19). 21 Herpesviridae Herpesviridae is a family of DNA viruses known to become latently present in different subsets of differentiated cells. There are three different subfamilies of Herpesviridae capable of infecting humans: alpha Herpesviridae, including herpes simplex virus (HSV) -1, HSV-2 and varicella zoster virus (VZV); beta Herpesviridae, including human cytomegalovirus (HCMV), human herpesvirus (HHV) -6A and HHV-6B, HHV-7; gamma Herpesviridae, that include Epstein-Barr virus (EBV) and Kaposi sarcoma associated herpesvirus (20). The most relevant virus of the Herpesviridae family will be next briefly described. Cytomegalovirus Human infection with cytomegalovirus (HCMV) is complex since it is difficult to have a clear epidemiologic profile of this infection. In most instances, primary HCMV infection is asymptomatic. HCMV infection invariably becomes chronic in the infected host, with viral latency and gene expression occurring persistently at low levels. Moreover, there is no vaccination implemented against this viral infection. Together, this helps to explain why the seropositivity is so high, ranging from 45% to 100% IgG seropositivity for the virus in different regions of the world (21,22). In Portugal, the 2001-2002 second National Serological Survey showed a seropositivity of 72.3% in men and 80.2% in women (23). Figure 3. Mechanisms of immunosuppression during CMV infection. Extracted from (24). HCMV evolved together with the human being for millions of years. It shows specificity to the species and adapted itself to override the host immune response. It encodes several proteins in its 22 genome that can evade the host defense. Metanalysis show that anti-CMV prophylaxis in transplantedorgan recipients is linked to decreased incidence of opportunistic infections. CMV is thought to induce immunosuppression of the host through a variety of mechanisms, including the induction of IL10 production and the suppression of antigen presenting cells (APCs), T cells and NK cells responses (Figure 3 and (24)). In young human adults, CMV-seropositivity is associated with improved antibody production, CD8+ T cell response and circulating levels of IFN-γ following anti-flu vaccination (25). Varicella Zoster virus VZV primary infection (chickenpox) occurs most often during infanthood, with up to 95% of adults over the age of 50 being IgG seropositive. In most cases, it resolves without sequelae. However, the virus can stay in the host in a latent state in the spinal and cranial sensorial ganglia. Later in life, and particularly in immunocompromised patients, Varicella Zoster reactivation can occur (shingles). Between 25-30% of all adults are at risk of developing secondary Herpes Zoster, with people over the age of 80 having a 50% risk of developing viral reactivation (26). Epstein-Barr virus EBV is the etiological agent of infectious mononucleosis. This is a relatively benign and selflimiting disease. However, the virus is not fully cleared in most individuals, persisting in in the cell nuclei of oropharyngeal epithelial cells and in memory B cells (27). Moreover, it is estimated that 2030% of adults are shedding live viral particles at any given time (27). In addition, the virus can live outside the human host for a considerable time. Hence, it is very contagious, with some estimates considering that up to 95% of the human population is seropositive (27). In most cases, this represents no threat to the host. Yet, in some individuals it can reactivate in later periods of life, especially those immunocompromised. Of note, this was also the first virus associated with malignant tumors in humans, when virus particles were first collected from jaw sarcomas tissues (27). Herpes simplex virus I and II In the human species, there are two strains of HSV, HSV-1 and HSV-2. It is estimated that 67% to 90% of the world population is infected with either of the two viruses. HSV-1 is the most frequent, with 3.7 billion people below the age of 50 living with the virus, either in its active or latent stage. For 23 HSV-2, it is estimated that 417 million people are living with the infection. In most cases, symptoms are minor and self-limiting. Yet, particularly HSV-2 is linked to malignant tumors (28). HSV downregulation of mucosal immunity of the vagina and infection reactivation are known to be associated with increased rates of HIV infection (29,30). Also, viral reactivation in mucosal tissues results in local recruitment of activated CD4+ T cells, possibility facilitating HIV transmission (31). 24 Viral co-infections During our life, we can be infected with multiple different viruses. In fact, co-infection may be the real-world scenario in which most infections develop. Yet, we have limited knowledge on how our immune system handles multiple simultaneous infections. There are many confounding factors that limit scientific research in this area. For instance, the study subjects may be infected with pathogens other than those we are evaluating. It can even occur the pathogens with which they are infected cannot be readily recognized by the diagnostic tools available at that time. It is also difficult to pinpoint precisely at what time one was infected with each pathogen; each infection may modulate, positively or negatively the clinical course of the concurrent infections, which does not facilitate epidemiologic investigation (32). HIV infections concurrent with hepatitis virus infections, either HBV or HCV, are the most studied viral co-infections occurring in humans. It is currently known HIV infection accelerates the development of clinical complications of hepatitis infections, including liver failure (33). On the other hand, hepatitis virus infections contribute to slower immunological recovery, including CD4+ T cell counts, in HIV infected individuals (34). 31 2002;20(SUPPL. 5):33–5. 18. Majda-Stanislawska E, Bednarek M, Kuydowicz J. Immunogenicity of inactivated hepatitis A vaccine in children with chronic liver disease. Pediatr Infect Dis J. 2004;23(6):571–4. 19. Wiedmann M, Liebert UG, Oesen U, Porst H, Wiese M, Schroeder S, et al. Decreased immunogenicity of recombinant hepatitis B vaccine in chronic hepatitis C. Hepatology. 2000;31(1):230–4. 20. Weidner-glunde M, Kruminis-kaszkiel E. Herpesviral Latency — Common Themes. Pathogens. 2020;9(125):1–26. 21. Patro ARK. 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J Virol. 33 2001;75(23):11392–400. 50. Stelekati E, Shin H, Doering TA, Dolfi D V., Ziegler CG, Beiting DP, et al. Bystander chronic infection negatively impacts development of CD8+T cell memory. Immunity. 2014;40(5):801– 13. 51. Clute SC, Watkin LB, Cornberg M, Naumov YN, Sullivan JL, Luzuriaga K, et al. Cross-reactive influenza virus-specific CD8+ T cells contribute to lymphoproliferation in Epstein-Barr virusassociated infectious mononucleosis. J Clin Invest. 2005;115(12):3602–12. 52. Ng CT, Snell LM, Brooks DG, Oldstone MBA. Networking at the level of host immunity: Immune cell interactions during persistent viral infections. Cell Host Microbe. 2013;13(6):652–64. 34 CHAPTER II. The CL13 mouse model of chronic infection The persistence of a clinically silent viral infection in mice was first reported in 1935, by Erich Traub (1). In his work, he injected naïve mice intracranially with a preparation containing smashed organs from mice apparently clinically well and symptom free. Following infection, previously naïve mice showed neurological clinical symptoms, including somnolence, photophobia, tremors of the legs and tonic muscle spasms (1). With this, Traub hypothesized that donor mice should be infected with an unknown virus, able to persist in the host for long periods of time without overt symptomology. Later, Traub identified this virus as lymphocytic choriomeningitis virus (LCMV), which naturally infects mice vertically in utero or shortly after birth, without clinical signs of infection in most mice, yet persisting at high viral titers in several organs in many animals (2,3). This model was insufficient to replicate viral infections in humans that install later in life such as cytomegalovirus (CMV) or hepatitis C virus (HCV). The team of Michael Oldstone first described a mouse model of chronic infection of LCMV with adulthood onset (4). Clone 13 (CL13) is a viral strain of LCMV that was originated from the parent virus strain LCMV-Armstrong (Arm) (4). The CL13 strain was first isolated from 2 months old BALB/C WEHI carrier mice, infected at birth with Arm (4). CL13 differs from the parent virus Arm in 5 nucleotide, that result in two amino acid substitutions in the viral glycoprotein (GP) (phenylalanine to leucine, F→L, GP260) and in the polymerase (lysine to glutamate, K→E, L1079) (5–7) that promotes extensive proliferation within dendritic cells (DCs) and fibroblastic reticular cells (8,9). In contrast to Arm, CL13 produces chronic infection with persistent viremia in blood and several organs, and impaired cytotoxic T lymphocytes (CTL) response for several months (4,10). 35 Establishment of chronicity and innate immunity The innate immune response is pivotal in the early control of Arm or CL13 infection. The higher levels of replication within DCs allows for increased antigen presentation to T cells while altering the homing of multiple immune players (11). This coincides with an increased peak of interferon (IFN)-I production compared to Arm (12). Simultaneously, the inhibitory programmed deathligand 1 (PD-L1) is upregulated in DCs and there is higher production of interleukin (IL) 10 (13). Together, this contributes to reduced T cell effector activity. The elevated infection of fibroblastic reticular cells results in severe, long-lasting, disruption of the fibroreticular mesh of several lymphoid organs, including the spleen (9,14). Moreover, CL13 results in increased expression of PD-L1 in the fibroblastic reticular cells, which further contributes to impair the cytotoxic activity of CD8+ T cells, disturbs the production of chemokine ligand (CCL) 19 and CCL21, important for T cell recruitment, and IL-7 production, which provides survival signals to T cells (9). When CL13 infection is established, there is also extensive deletion of several clones of T cells. It is hypothesized that natural killer cells (NK cells) may have a role in this process. Following rapid NK cell differentiation and activation in the early phases of CL13 infection, NK cells show extensive changes in their phenotype and apparently contribute to the killing of CD4+ T cells, hence impairing CD8+ T cell activation and differentiation (15,16). How each of these innate immune players contributes to the establishment of chronic CL13 infection is described below. 36 Dendritic Cells CL13 also replicates to higher levels than Arm in plasmacytoid DCs (pDCs) (8,17). CL13 infections results in higher levels of infection of antigen presenting cells (APCs) in the white pulp of the spleens (18). Splenic DCs have high levels of expression of α-dystroglycan (α-DG) which serves as a receptor for LCMV entry into cells (17,19). Interestingly, LCMV strains that produce chronic infections, such as CL13, bind strongly to α-DG, whereas the acutely resolved Arm does not. This is particularly evident in the white pulp of the spleen, where high infection rates of DCs are observed (19). This enhances killing of DCs mediated by CD8+ T cells (18). Therefore, as CL13 infection progresses into chronicity, the number of DCs decreases (12). Furthermore, the remaining DCs bear an exhausted phenotype and respond poorly to stimuli (12,20). The remaining DCs localize along the red pulp and the marginal zone and produce high levels of IL-10. As the infection progresses, they become more disperse, co-localizing with CD4+ T cells and B cells (17). In addition, during CL13 infection, DCs produce a wide range of cytokines, including IFN-I, IL-10, IL-12, tumor necrosis factor (TNF) , IL-1α, and chemokines, such as CCL2, CCL4, and CCL5 (17,21,22). suggesting a role for DCs in the regulation of CD4+ T cells and B cells (23). 37 Interferon-I IFN-I signaling is pivotal in the establishment of CL13 chronic infection and arguably one of the most debated figures in the field (24). Infection with LCMV induces IFN-I (both IFN- and IFN- ) production, mostly by pDCs, that peaks at 12-48 h of infection (Figure 6 and (21,22)). Absence of IFN-I signaling in this period results in increased LCMV viral loads in the spleen of infected mice (22). Figure 6. IFN-I Splenic levels of IFN-I mRNA over time after infection with Arm or Cl13. Extracted from (12). Results are expressed as the difference between the Ct values of the housekeeping gene HPRT and that of the target gene of interest (ΔCt HPRT-target) in each sample. Following the initial peak of IFN-I, the levels of IFN-I bioactivity and of RNA transcripts start to decrease, reaching values that may be lower than in non-infected mice (12). This reduction in IFN-I production is difficult to revert by different additional stimuli, including TLR ligands or secondary 38 unrelated infection (25). Surprisingly, the activity of IFN-I persists far longer throughout CL13 infection. In fact, at one month post infection, the expression of several IFN-I receptor inducible genes including Mx2, Oas1a, Oas1g, Oas2, Oas3, and others remains increased in CL13 infected mice (21). Current evidence suggests IFN-I production during CL13 infection occurs following the activation of the transcription factor (TF) TLR7 which promotes the transcription of IFN-I genes (22,23). The activation of other pathways, via TLR3 or TLR9 ligands, also results in increased IFN-I production (12,20,26–28). There is also evidence that the recognition of viral RNA by the MDA5 RNA sensor is also important for the activation of the TLR7 pathway (22). Figure 7 IFN-I role in clearance/ persistence of LCMV infection. Extracted from (1) In the LCMV WE acute infection model, another LCMV virus strain that results in acute infection, used by many research labs, IFN-I signaling has a preponderant role in the clonal proliferation of LCMV specific CD8+ T cells. When transferring LCMV specific P14 T CD8+ T cells into LCMV WE infected recipients, they proliferate extensively, making up to 60% of CD8+ T cells in the recipients around 10 days post infection (dpi), with increased IFN-γ production and expression of the maturation markers KLRG1, CD62L and CD44. In opposition, Ifnar1 -/- P14 T cells do not proliferate and have a severely reduced IFN-γ response, with decreased maturation. This indicates that IFN-I signaling is required for the development of a CD8+ T cell response to LCMV (22,29). Interestingly, the impaired proliferation of transferred Ifnar1 -/- P14 T cells appears to be infection specific, because they proliferate 39 and expands normally Ifnar1-/- in mice infected recombinant vaccinia virus carrying the GP33 epitope (29). Oxenius and colleagues showed that IFN-I signaling protects LCMV specific P14 T cells from the action of NK cell mediated killing (30). Transferred P14 Ifnar1-/- T cells are preferentially killed by NK cells, compared to the WT counterparts (30). Interestingly, this appears to be dependent on NKp46 signaling in NK cells, because similar to after NK cell depletion, the preferential killing of Ifnar1 -/- P14 cells does not occur in Ncr1icre/icre mice, which lack NKp46 (31). Moreover, Diefenbach and colleagues showed that P14 T cells co-cultured with Prf-/- NK cells, lacking perforin production, are not preferentially killed when compared to the WT counterparts (32). Diefenbach and colleagues also suggested the possible mechanism of protection induced by IFN-I signaling in T cells. A micro-array of isolated WT T cells compared with Ifnar1-/- T cells, shows that IFN-I signaling upregulates several MHC-I molecules that bind to several inhibitory receptors on NK cells (32). Considering that most of the early IFN-I comes from DCs, one would imagine that the absence of this source of IFN-I would lead to a severe impairment of the CD8+ T cells response. Yet, this is not the case. The IFN-I produced by DCs has a limited impact in the CD8+ T cells response to LCMV (17). In addition to this, infected CD8- DCs alone are unable to stimulate LCMV-specific CD4+ and CD8+ T cell proliferation (8). Furthermore, their absence does not translate into lower levels of LCMV-specific CD8+ T cell response (22). As CL13 progresses into chronicity, IFN-I signaling also contributes to the reduction in the number of pDCs, because it reduces pDCs precursors in the BM in a STAT2-/-, but not STAT1-/- depended way (20,33). Therefore, blocking IFNAR signaling increases BM precursors, to numbers similar to those observed in non-infected mice (20). Yet, it does not restore the numbers of splenic pDCs (20). Current evidence suggests that IFN-I signaling is also required for the proliferation of pDCs (20). In this process, IFN-I induces TLR7 signaling and stimulates the proliferation of pDCs in a cellintrinsic way (20). However, this catalyzes a negative feedback mechanism in which IFN-I production is suppressed and pDCs become unresponsive to new stimuli (20). IFN-I signaling likely also contributes to increased expression of immunosuppressor mediators. In CL13 infected Ifnar1 -/- mice, the expression of PD-L1, the ligand for the inhibitory receptor Programmed cell death protein 1 (PD1) in DCs and macrophages, is significantly decreased. Infected Ifnar1 -/- mice also show a markedly decreased production of IL-10. The same is observed when 40 blocking IFNAR with a blocking antibody. Interestingly, the absence of IFN-I signaling, during the establishment of chronic CL13 infection, either by absolute absence, as is the case of Ifnar1 -/- mice, or reduced activity using blocking antibodies, results in increased viremia. These observations suggest that IFN-I has an important role in controlling infection. However, by 15 dpi with CL13, the effect of blocking IFNAR is attenuated, with no changes in IL-10 production. In the long term, blocking IFNAR signaling may contribute to improved viral control, with improved APC activity of DCs, that henceforward express PD-L1 in similar levels to naïve mice (21). It is yet to be explained why IFN-I production relates to chronicity of CL13 infection when its levels progressively decrease as the infection progresses (Figure 7 summarizes the main key points of this debate). To answer this, Oldstone and colleagues infected mice in which either the IFNAR receptor or IFN-β were blocked: In mice where the IFNAR receptor was blocked, LCMV early replication was increased, with higher rates of infection of DCs and monocytes. However, this did not occur in mice were IFN-β was blocked. Moreover, blockade of IFN-β did not result in an increased inflammatory response in the spleen. Also, when blocking IFNAR, there was increased inflammation of the spleen. Intriguingly, blocking IFN-β translated into improved specific T cell response and decreased viral titers, similarly to when blocking only IFNAR. Therefore, we may conjecture that IFN-β contributes to decreased pathology early in LCMV infection but promotes chronicity in the long term due to the inherent reduction of the T cell responses (34). 47 CD8+ T cells Figure 10. Establishment of CL13 chronicity and CD8+ T-cell exhaustion. Extracted from (47). The epitope-specific CD8+ T cell response to CL13 differs from that to Arm (10). For CL13, CD8+ T cells specific to some LCMV epitopes are eliminated, while others maintain an exhausted phenotype throughout the infection, with increased expression of CD44 and CD69, and decreased expression of CD62L, but without antiviral effector activity (52). The reason for the persistence of some exhausted CD8+ T cells clones and the deletion of others has been strongly debated (Figure 9 and 10). Ahmed and colleagues suggested that the presence of continued antigen presentation leads to deletion of a significant part of the antigen specific CD8+ T cells and exhaustion of the remaining cells (53). In mice that recover from acute Arm, a pool of LCMV specific CD127hi, CD122hi memory CD8+ T cells remains long after infection resolution (54). In contrast, in chronically CL13 infected mice, this same LCMV-specific population has reduced expression of both markers (54). Moreover, and as exhaustion is being established, the production of IL-2, TNF-α and IFN-γ in CD8+ T cells progressively decreases. Also, CD8+ T cells gradually decrease polyfunctionality, motility and metabolic activity (10,55–58). Even if stimulated with adjuvants such as IFN-I or TLR7 ligands, in CL13 infected mice, the CTL response is small (12). Interestingly, suppression of cytolytic activity in exhausted CD8+ T cells 48 is not as startling as it is with the other effector functions (59). This suggests that some CD8+ T cell activity remains and exerts residual virus control. The expression of inhibitory receptors in CD8+ T cells is directly correlated with the establishment of CD8+ T cells exhaustion (56). During CL13 infection, CD8+ T cells express multiple inhibitory receptors, including PD1, LAG-3, CD160, 2B4, CTLA-4, PIR-B, GP49, KLRG1, NKG2A, Tim-3, BTLA and TIGIT (56,60–63). Moreover the differentiation of effector CD8+ T cells into memory cells is impaired, with reduced expression of CD62L, CD27, and CXCR3 (64). Interestingly, as chronicity is established, the vast majority of CD8+ T cells express at least 3 of these receptors (60). Between 65% to 80% of LCMV specific CD8+ T cells co-expressing Tim-3 and PD1, while producing the immune suppressor IL-10 (61). Notably, the larger the number of inhibitory receptors that CD8+ T cells express during CL13 infection, the lower the IL-2, TNF-α and IFN-γ production (60,61) This exhaustion phenotype of CD8+ T cells can be somewhat reverted by blocking PD1 signaling. Blocking PD-L1 or PD1 decreases the exhaustion of CD8+ T cells, which show increased proliferation and polyfunctional activity, with improved TNF-α and IFN-γ simultaneous production, increased degranulation and CD107 expression, increased metabolic activity and motility, and results in improved viral control (57,58,60,63). Blockade of PD1 signaling results in TF T cell factor 1 (TCF 1) dependent proliferation of PD1+ CD8+ T cells. These cells co-localize with naïve T-cells in lymphoid tissues and bear a gene profile that resembles stem cells and express several co-stimulatory molecules, including ICOS and CD28 (65,66). In contrast, the blockade of LAG-3, Tim-3 or TIGIT alone does result in improved viral control or T-cell function, yet it potentiates the effects from PD1 blockade (60–62,67). Interestingly, the effects of PD1 blockade therapy may be absent if stimulating other inhibitory receptors simultaneously. When blocking Tim-3 alone, that does not result in increased exhaustion of CD8+ T cells but is enough to impair the response to PD1 blockade therapy (68). Considering the sharp exhaustion phenotype associated with the expression of inhibitory receptors, it is possible to hypothesize that impeding the expression of such receptors early in infection, CD8+ T cell exhaustion would be averted and CL13 persistence avoided. However, CL13 infection is lethal to PD-L1-/- due to the severe immunopathology (63). This suggests that the expression of inhibitory receptors is necessary to protect the infected mice from CD8+ T cell cytotoxicity during persisting infections such as CL13. PD1-/- CD8+ T cells transferred to C57BL/6 mice proliferate more 49 and show improved cytotoxicity between 8-14 dpi with CL13 yet later become even more exhausted than the host CD8+ T cells (69), suggesting that other mechanisms counteract the absence of PD1 signaling to induce of CD8+ T cell exhaustion. CL13 infection can suppress the CD8+ T cell response by inducing the death of virus-specific cells. Decaluwe and her colleagues showed that during CL13 infection, the differentiation marker CD122 is significantly increased, coinciding with other classical exhaustion markers CD160, 2B4, LAG3, TIM-3 and PD1. Interestingly, those cells expressing higher levels of PD1 are also the ones expressing the higher levels of the death markers 7AAD and annexin V. Not all remaining CD8+ T cells become exhausted during chronic CL13 infection. Hasenkrug’s team described a population of CD8+ T cells that expresses the inhibitory receptor SIRP (70). Surprisingly, in this subset of cells, the expression of multiple inhibitory and stimulatory receptors is increased, including CD122, Tim3, Lag3, CD95, CD43, CD44, CD40, CD728, KLRG1, CD62L, CD47 and CXCR31(70). Also, the SIRP+ CD8+ T cells have increased proliferative capability, IFN- production and cytolytic activity than the SIRP- counterparts (70). Oldstone and colleagues studied the early suppression of CD8+ T cell response upon CL13 infection. When wild-type mice were primed with Arm up to 8 hours prior to CL13 infection, chronicity did not occur. Instead, GP33 specific CD8+ T cells were fully functional, with increased IFN-γ and TNF-α production. Moreover, NP396 specific CD8+ T cells, which, typically become virtually undetectable as CL13 infection becomes chronic, were increased and functional (71). During chronic infection, CL13-specific CD8+ T cells respond poorly to proliferative stimuli. 30 dpi with CL13, CD8+ T cells proliferated poorly following in vitro IL7 and IL-15 stimulation, whereas those from Arm infected mice proliferated extensively (72). Interestingly, exhausted CL13 specific CD8+ T cells can partially respond to a secondary Arm infection. When CL13-specific CD8+ T cells from mice chronically infected with CL13 are transferred to naïve mice, which are subsequently infected with Arm, they expand at similar levels as virus-specific CD8+ T cells isolated from Arm infected mice. Yet, CD8+ T cells from chronically infected mice show reduced cytokine production, including TNF-α, and retain high PD1 expression (73). This suggests the exhaustion of CD8+ T cells persists even in daughter cells, which implies the differentiation process of CD8+ T cells is irreversibly altered with prolonged virus exposure and antigen overload. 50 The establishment of exhaustion of CD8+ T cells in chronic LCMV infection is also regulated by several TFs. Wherry and colleagues observed that in CL13 infected mice, virus-specific CD8+ T cells overexpress Blimp-1, a transcriptional repressor of IFN-I related genes. Moreover, the expression of this repressor gradually increases as the infection progresses. The expression of Blimp-1 is also associated with the expression of the inhibitory receptors PD1, LAG3, CD160, and 2B4. When conditionally eliminating Blimp-1 in CD8+ T cells, the expression of those inhibitory receptors was significantly decreased. Moreover, those CD8+ T cells expressed a more memory-like phenotype, with increased levels of CD127 and CD62L. Ultimately, the expansion of LCMV specific cells was significantly improved (74). Blimp-1 expression is also more abundant in exhausted CD8+ T cells that have low expression of the TF TCF-1. Interestingly, TCF-1 overexpressing P14 CD8+ T cells persisted much longer and showed a less exhausted phenotype than the C57BL/6 counterparts, including decreased Blimp-1 expression. Therefore TCF-1 expression is associated with increased frequencies of memory-like CD8+ T cells, that have reduced signs of suppression and exhaustion and improved effector activity, with increased GzmB production (75,76). Many other TFs are important for the establishment of CD8+ T cell exhaustion. For instance, PD1 expression requires the modulation by the TF FoxO1 (77). T-bet is more associated with the differentiation of effector CD8+ T cells following acute infection, whereas EOMES is more associated with exhausted CD8+ T cells (78). The tumor suppressor VHL negatively downregulates HIF expression, decreasing CD8+ T cell effector functions but protecting chronically infected mice from increased CD8+ T cell induced immunopathology. VHL-/- CD8+ T cells have reduced expression of the inhibiting receptor KLRG1, expand to higher numbers in infected mice and express increased levels of GzmB and other activation markers (79). Absence of the TFs IRF4 or BATF results in limited CD8+ T cell responses in mice infected with with 2x106 PFUs of LCMV WE (80). In contrast, work by Kallies and colleagues showed that exhausted CD8+ T cells have increased expression of IRF4 and BAFT. Compared to WT P14 cells IRF4+/- P14 cells transferred into LCMV infected mice were more poly-functional and had decreased expression of several inhibitory receptors, including PD1, TIGIT, Tim-3, Lag3, 2B4 and CTLA-4 (81). Together, these results suggest that CD8+ T cells require optimal levels of IRF4 and BAFT to overcome exhaustion during the early and later phases of LCMV infection. In addition, the TF NFAT promotes CD8+ T cell exhaustion but also effector phenotype when interacting with the TF AP-1 (82). 51 Figure 11. LCMV-depended antigen stimulation and CD8+ T cell differentiation. Extracted from (72). With the establishment of chronicity, the continued antigen overload of CD8+ T cells appears to suppress the progression of effector CD8+ T cells into the memory pool, possibly resulting in reduced life span (Figure 11). Ultimately, this results in fewer and less responsive memory cells. When LCMVspecific CD8+ T cells, obtained from CL13 infected mice at 8 dpi, were transferred into mice that had been infected with Arm also for 8 days, were still present at 30 dpi and expressed CD127 (83). This suggests that in the early phase of CL13 infection, virus-specific CD8+ T cells retain the potential to become memory cells. Yet, when the LCMV-specific CD8 T cells were obtained from mice infected with CL13 for 30 days, they persisted at lower levels than in Arm infected control mice, with reduced upregulation of CD127, indicating they were unable to survive and become protective memory cells (83). 52 CD4+ T cells CD4+ T cells are pivotal in controlling LCMV infection and provide crucial help for CD8+ T cells responses (Figure 12) by inducing IL-12 production in DCs through CD40L-CD40 interactions IL-12. In addition, CD4+ T cells produce IL-2, which promotes CD8+ T cell survival (84,85). During LCMV infection, the CD4+ T helper response is dependent on IL-6 signaling. IL-6-/- mice have persistent and increased serum viremia (86). Absence of IL6 signaling, including in a CD4+ T cell restricted manner, results in reduced numbers of GP66 virus-specific CD4+ T helper cells and decreased production of IFN-γ and IL-21 in a per cell basis (86,87). In the absence of IL-6 signaling, there are also reduced numbers of GP33-specific CD8+ T cells and persistent serum viremia and increased viral loads in several organs, including the spleen, the liver and the lungs (87). In vitro stimulation of LCMV-specific SMARTA CD4+ T cells with IL-6 resulted in upregulation of STAT1 and STAT3 and increased production of IL-21. Together, this suggests that IL-6 stimulates T helper cells, via the activation of STAT1 and STA2, with increased production of IL-21, which promotes the expansion of virus-specific CD8+ T cells. In the absence of CD4+ T cells, LCMV-specific CD8+ T cell clones expand at reduced levels and show a more exhausted phenotype, with reduced cytotoxic activity and potential to respond to new viral stimuli, resulting in increased CL13 loads and impaired viral control (52,55,60,88,89). Transfer of LCMV-specific SMARTA CD4+ T cells into CL13 chronically infected mice rescues exhausted CD8+ T cell effector function, proliferation and cytokine production, including IFN-γ, leading to improved viral control, with reduced serum LCMV viremia (90). Together, this clearly shows a role for CD4+ T cells in the modulation of CD8+ T cells following viral infection. Similarly to CD8+ T cells, CD4+ T cells progressively lose effector activity and become exhausted with the establishment of chronic CL13 infection. In CL13 chronically infected mice CD4+ T cells show reduced production of IL-2, TNF-α and IFN-γ (91,92). Moreover, GP66 virus-specific CD4+ T cells increase expression of several inhibitory receptors, including PD1, LAG-3 and CTLA4, with a markedly exhausted molecular signature (92). 53 Figure 12. CD4+ T cell help for CD8+ T cell differentiation in LCMV infection. Extracted from (93). Interestingly, a population of CD4+ T cells persists during infection and shows increased IL-10 production and FoxP3 expression, as well as CTLA-4, CD39, GzmB, ICOS, PD1. PD-L1, CD44 and CD69, which suggests they bear a regulatory phenotype (91,92,94). Using Foxp3DTR mice, Ahmed and colleagues demonstrated a role for regulatory T cells in suppressing CD8+ T cells (94). In mice, after ablation of regulatory T cells in Foxp3DTR, LCMV specific CD8+ T cells displayed increased expression of the proliferation marker Ki67 and were highly activated, with increased expression of CD127, CD44 and GzmB (94). Moreover, following ex vivo stimulation with multiple LCMV peptides, they produced 54 larger amounts of IFN- (94). Yet, the ablation of regulatory T cells alone did not result in improved viral control (94). This was possibly linked to continued PD1 expression in CD8+ T cells. Hence, when blocking PD1 and ablating regulatory T cells, there was improved viral control, comparing to any of the two alone (94). Altogether, these data suggest that with the establishment of chronic CL13 infection, a population of CD4+ regulatory T cells emerges, with high levels of IL-10 production, and actively suppresses CD8+ T cell effector function. Figure 13. Drivers of T cell exhaustion in CL13 infection. Extracted from (95). In summary, CL13 infection produces an immunosuppressed state within the host, with many CD8+ T cell clones deleted or exhausted. The many possible reasons for this to occur, are shown in Figure 13, including alterations in APCs, the structure of the lymphoid organs, pro and antiinflammatory cytokines, as well as changes in CD4+ T and NK cells. Despite being detrimental for virus control, this immunosuppression may be beneficial to the host. It is hypothesized that without exhaustion of T-cells, the immune response of the host would be so strong that could lead to death (95). 55 B cells Many virus-specific B cells are deleted at the onset of CL13 infection, showing increased incorporation of 7AAD and surface expression of Annexin V (96). Also, during infection with LCMVDOCILE, another LCMV virus strain that results in chronic infection, there is significant impairment of the neutralizing antibody response, even in the setting of an unrelated subsequent infection with Vesicular stomatitis virus – New Jersey strain (VSV-NJ) (97). Lamarre and colleagues described a similar outcome following CL13 infection, with reduced specific IgG response to CL13 (50). Moreover, as the infection progresses, the B cell numbers are reduced, together with a disrupted spleen architecture (50). Deletion of B cells is time dependent and likely related to the peak of IFN-I that follows shortly after the onset of CL13 (50,96,98). IFN-I probably acts on B cells early during the infection because at 1-week post infection, the viral loads are high in CL13 infected mice but undetectable in Arm infected mice. Despite current conflicting evidence, it is likely that IFN-I signaling acts indirectly on B cells, modulating other immune cells that respond to CL13 infection, but also directly, likely via modulation of interferon stimulated genes (ISGs) (50,96,98). This hypothesis still requires further studies to be fully uncovered. 56 References 1. Murira A, Lamarre A. 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Eur J Immunol. 2016;1–13. 63 CHAPTER III. The mousepox mouse model of acute infection Variola and smallpox The eradication of smallpox is one of the greatest achievements of public health. This was a disease that affected humankind for thousands of years. The first biological evidences of the disease were collected in ancient Egypt mummies from the 3rd century BC. The first written reports of the disease only emerged much later in the 4th century AD (1). Smallpox was caused by the variola virus. Variola virus is a poxvirus from the Poxviridae family, subfamily Chordopoxvirinae , genus orthopoxvirus (OPV). There are several other orthopoxviruses, such as vaccinia virus, cowpox virus, monkeypox viruses and other serologically cross-reactive animal-borne viruses (2,3). OPVs are amongst the largest viruses infecting mammals in nature, measuring 200 x 400 nm and show a brick-like structure. OPVs have and envelope that encases the other viral structures: an outer lipidic membrane; several lateral bodies, which contain several enzymes required for hijacking the infected cell; and the core membrane, surrounding the viral double-stranded, linear DNA and all the proteins necessary for mRNA synthesis to occur in the infected cell cytoplasm (2,3). Infections with Poxviruses occur through 2 main routes: respiratory and contact trough skin abrasions. From the local of entry, the virus proliferates and propagates rapidly reaching the draining lymph nodes (dLN). In the case of smallpox, a short period of viremia is followed by a low-symptomatic incubation period in which the virus is mostly restricted to the reticuloendothelial system. After 4 to 14 days, a new period of viremia occurs, and the virus propagates to the mucous membranes in the mouth and pharynx and the capillary epithelium of the dermal layer of the skin. During this prodromal phase, patients abruptly develop severe headaches and backaches and become feverish, with temperature rising over 40 ºC. Concomitantly, they show severe enanthem over the tongue, mouth, and oropharynx. Two days later, the infected individuals develop the characteristic rash lesions, first in body extremities and then throughout the whole body. This is the period in which infected individuals are the most contagious, with large amounts of viable virus being shed in urine and other body secretions. At this stage, the virus is also present in the spleen and liver, lymph nodes, bone marrow, kidneys and other viscera. The ablation of the infection is mostly cell-mediated, with macrophages, cytotoxic T cells and B cells taking major roles. However, up to 30% of the virus genome encodes for 64 proteins which functions are to evade and counteract the immune response of the host. Death from smallpox occurs in 1/3 of the infected individuals and is associated with toxemia from immune complexes and hypotension (3). In the late XVIII century, Edward Jenner pioneered the discovery of the first effective vaccine against smallpox, simultaneously the first reported vaccine in the history of mankind. Jenner observed milkmaids who milked cows affected with peculiar eruptions on their teats, would contract a relatively benign disease, cowpox and possibly eliciting protection from mousepox. Jenner confirmed this association and developed a protocol, ethically questionable by current standards, of inoculating children in succession with a variant of cowpox and then exposing them to smallpox, to which they all resisted (4–6). From that time onwards, this new strategy was widely adopted to eradicate smallpox, but that was only attained during the XX century, when the joint efforts of the WHO and thousands of public health organizations around the globe drove the eradication of the disease. From the last reported case in Somalia, in 1977, to the official declaration of eradication in 1980 and cessation of widespread public vaccination, today there are only two reported stocks, both at WHO facilities, in Atlanta, USA, and in Novosibirsk, Russian Federation (1).Yet, the risk of biological warfare and climate change, which is exposing contaminated biological tissues from permafrost grounds, still threatens the great achievement of smallpox eradication (1,7). 65 The mousepox model of acute infection Ectromelia virus (ECTV) is the agent of mousepox, an acute lethal viral disease of the mouse, very similar to human smallpox. It was first suggested as a model to study human smallpox since it is closely related to the variola and vaccinia virus (8). Besides being the optimal mouse-specific model to study the human-specific variola infection, ECTV infection model provides an excellent model to study other viruses that spread lymphohematogenously, including those that responsible for smallpox, chickenpox, West Nile fever, Dengue fever, yellow fever, rubella and measles (9). In the mouse, ECTV infection occurs in the skin of the footpad, trough skin abrasions (10). From there, the virus proliferates and propagates reaching the dLNs. Following 2-3 dpi with ECTV, the virus spreads lymphohematogenously to the liver and spleen, the primary infection sites of ECTV. By 4 dpi it is already possible to detect placable ECTV from the spleen and liver (11). Following footpad infection, Mice can be either genetically susceptible and succumb to mousepox or resistant to the disease. BALB/C, DBA/2J and A/J mice succumb following ECTV infection, whereas C57BL/6J and 129 mice recover from disease (12). In resistant strains, virus loads peaks in the footpad and dLNs at 5-6 days following infection, and at 7 dpi in the liver and spleen. After that, most resistant strains show a progressive decrease in viral load, that is mostly undetectable following 10 dpi with ECTV, whereas susceptible strains continue to have progressively higher viral loads, until the moment they succumb to the disease (12), arguably due to liver necrosis (9). 66 Figure 14. The pathogenesis of mousepox. Extracted from (11). 67 Figure 15. Clinical features and ECTV virus load in organs during mousepox in resistant mice. Extracted from (11). 68 Innate immunity to ECTV The immune response to ECTV starts right in the footpad skin. Infected MHC-IIhi DCs rapidly migrate from the footpad skin to the dLN, where their numbers and response peak at 24 post-infection. These DCs upregulate the expression of CCL2, CCL7, CXCL9, several interleukins, including IL18, TNFα and IFN-I. Together, this contributes to the accumulation of iMOs and NK cells in the dLN (13). iMOs are able to modulate the recruitment and activation of several immune players, including NK and T cells, as they are major sources of IFN-I and CXCL9 (9). NK cells can modulate the recruitment and activation of several immune cells, including T cells, as well as acting directly in eliminating infected cells. The detail of some of these pathways is detailed ahead. IFN-I IFN-I stands out as one of the most prominent players of the innate immune response in protection from mousepox. Depletion of IFN-I signaling in C57BL/6 mice, using antiIFN-I antibodies, results in increased mortality following ECTV infection, with faster viral spread from the dLNs to the liver and spleen, and overall higher ECTV viral titers (14). Similarly, the absence of Ifnar1 signaling in Ifnar1 -/- 129S7 mice results in full susceptibility to ECTV infection, whereas WT 129S7 mice are fully resistant to this infection (15). The role of IFN-I is more evident early in ECTV infection. Its levels increase at the dLNs as early as 1-3 dpi with ECTV. This IFN-I production is highly functional, promoting the expression of downstream interferon induced genes (ISGs), such as MX1, IRF-7, and ISG-15 (16). Interestingly, ECTV developed a decoy receptor for IFN-I that sequesters IFN-I molecules, likely impairing its bioactivity (17). When infection occurs with the ECTV-Δ166 strain, that does not express this decoy receptor, the EVM166 protein is no longer observable on the cell surfaces of infected cells, with overall improved viral control. Hence when infecting BALB/C mice with the ECTV-Δ166, the mice are fully resistant to infection, in stark contrast to what occurs following WT ECTV infection (18). 69 NK cells NK cells are lymphoid cells that, differently from T cells, do not bear antigen specific T cell receptors (TCRs). Instead, they express a pool of activating and inhibitory receptors that modulate their effector activity (Table 4 and (19,20)) Several cytokines modulate NK cell activation including TNF-α, IL2 +, IL12, IL15, IL18 and IFN-I (21–24). IL12 and TNF-α promote IFN-γ production (24). IL15 induces NK cell proliferation (24,25). IL2, IL12 and IL18 promotes the development of NK cytolytic activity (26,27). IL2, IL12 and IL18 can also promote NK cell suppressive activity and IL10 production (21,28–30). Other than cytokine receptors, NK cells have receptors that directly sense danger signals from infected cells, such as the homodimeric NKG2D receptor, which is upregulated in infection, as it was described in ECTV infection (31). NKG2D senses several ligands that are only expressed in infected cells, such as RAE-1, MULT-1 and several H60 proteins isoforms, all of them capable of inducing similar levels of cytotoxicity in NK cells (32). Blocking of NKG2D during ECTV infection impairs NK cells function and leads to increased viral loads and lethality (31). CD94 and NKG2A form a heterodimeric inhibitory receptor that binds to Qa-1b, a non-classical MHC I molecule, to restrain unwanted NK cell activation (33). CD94 also forms activating heterodimeric receptors, with NKG2E and NKG2C that also binds with Qa-1b. While CD94 is necessary for optimal NK cell mediated resistance to mousepox (34), NKG2C and E seem to be dispensable (Ferez-Ruiz, in preparation and (34)). In the mouse, there is also another family of activating and inhibitory receptors, the Ly49 family, absent in humans, that senses MHC-I receptors. Those with inhibitory functions include Ly49A, Ly49C, Ly49I and Ly49P. Most of them signal trough ITIM. Those with activating functions include Ly49D and Ly49H. Most of them signal through DAP12 (35–37). Ly49H is particularly interesting because it is the only one to which viral specificity is known. It binds specifically to m157, a viral glycoprotein encoded my MCMV, which in turn activates NK cells (37,38). After an early non-specific proliferation period, Ly49H+ NK cells expand and proliferate, generating a memory like population against MCMV, in what is suggested to be an adaptive immune response from NK cells (39,40). 70 Despite the discovery of antigen specificity in NK cells targeting MCMV, no other specific viral antigens that would activate or inhibit NK cells has been uncovered so far. Figure 16. NK cell mediated killing. Extracted from (41). NK cells act by killing the target cell (Figure 16). The main mechanism of NK cell killing is granule exocytosis whereby they release perforin, that open pores in target cells, and granzymes, which are serine proteases that induce apoptosis after entering the cytosol of the target cells through the pores generated by perforin (27,42). NK cells can also induce apoptosis of target cells via tumor necrosis factors (TNF), with the production of the death-receptor ligands FasL and TRAIL (41). The action of NK cells also involves the modulation of other immune cells. They produce several cytokines, including IFN-γ, TNF-α, GM-CSF and several chemokines, including CCL1, CCL2, CCL3, CCL4 and CXCL8, which modulate other innate and adaptive immune responses (43). NK cells can contribute to the suppression of immune responses, including with production of IL10 (21,44). 71 Table 4. Inhibitory and activating receptors in NK cells. Extracted from (37). NK cells are major innate immunity mediators in the response to ECTV infection. Their depletion in mousepox resistant C57BL/6 mice results in full susceptibility to disease (45). NK cells help control virus replication in the spleen and liver, without the help of T cells, up to 6 dpi with ECTV 72 (46). They are recruited to the site of infection by inflammatory monocytes, proliferate and produce IFNγ, eliciting an anti-viral state, upon surrounding non-infected cells, and producing granzymes, that directly kill infected cells in lymph nodes, liver, and spleen (46). Upon infection with ECTV, the number of NK peaks at 2 dpi in the draining lymph nodes and 56 dpi in the spleen and liver (46). After 6 dpi, the NK cell activity is no longer required for optimal recovery from mousepox, since their depletion does not result in increased mortality (31). The current understanding of NK cells shows that they help curb ECTV infection by exerting cytotoxic activity, through granule exocytosis and by producing IFN-γ. This IFN-γ production helps in promoting a pro-inflammatory state in the infection site that recruits more NK cells and promotes T cell activation (13,31,46). These functions require mostly the more mature NK cells. Sigal and colleagues showed that C57BL/6 aged mice, that succumb to mousepox, have decreased frequencies of the R3 (CD27-CD11b+) subset on NK cells, which is the most mature subsets and increased frequency of the more immature R1 (CD27+CD11b-) NK cell subset. This seems to be a broad defect in aged mice, as this phenotype is observable both in the spleen and in the bone marrow (25). 79 Part II – Results 80 CHAPTER IV. Chronic Lymphocytic Choriomeningitis infection causes susceptibility to mousepox and impairs Natural Killer cell maturation and function Pedro Alves-Peixoto 1,2,3, Maria Férez, Cory Knudson1, Colby Stotesbury1, Carolina R Melo-Silva1, Eric B. Wong1, Margarida Correia-Neves2.3 and Luis J. Sigal1,# Affiliations 1Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA, USA. 2Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, 4710-057, Portugal. 3ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, 4806-909, Portugal. DOI: 10.1128/JVI.01831-19 81 Abstract Introduction 82 83 Results 84 85 86 87 88 95 96 CHAPTER V. Role of IFN-I in NK cell maturation and activation during acute and chronic LCMV CL13 infection Running title: IFN-I signaling in NK cells during infection Pedro Alves-Peixoto 1,2,3, Carolina R Melo-Silva1, Luis J Sigal1,# Affiliations 1Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA, USA. 2Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, 4710-057, Portugal. 3ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, 4806-909, Portugal. (Manuscript in preparation) 97 Abstract The important role of Natural killer (NK) cells in the control of acute viral infections can become dysfunctional during chronic viral infections. Whether the Type I interferons (IFN-Is) IFN-α and IFN-β play a role in this dysfunction is unknow. Using the lymphocytic choriomeningitis virus clone 13 (CL13) model of chronic infection, we show that at 8 days post infection (dpi) with CL13, the large increase in the frequency of NK cells expressing the activation markers CD11b, GzmB, and KLRG1 was highly impaired in mice deficient in the IFN-I receptor (Ifnar1-/- ) while mice deficient in IFN-β( Ifnb1 -/-) had an intermediate phenotype. This suggests that IFN-α and IFN-β have a synergistic role during acute infection. In contrast, at 30 dpi with CL13, the maturation and activation defects of NK cells were more pronounced in Ifnb1 -/- than in Ifnar1-/- mice suggesting that during chronic infection IFN-β promotes maturation and activation and IFN-α restrains them. Our data also suggests that IFN-I is important to preserve late-stage GzmB production by NK cells. Altogether, our data suggests that IFN-α and IFN-β may have opposing roles during chronic infections and this may have implications for the use of IFN-I as therapies. 98 Introduction Natural killer (NK) cells are bone marrow-derived lymphocytes, critical in the innate immune response and surveillance of viral infections and cancer (1–3). When NK cells recognize infected cells or tumor cells, they promptly release cytotoxic mediators, such as granzyme B (GzmB) and perforins, and produce pro-inflammatory cytokines such as interferon-γ (IFN-γ) (1–3). The maturation of NK cells begins in the bone marrow and continues as they migrate to the periphery (4,5).(6,7). In the mouse, NK cells can be classified into four distinct maturation groups according to their expression of CD27 and CD11b. R0 (CD27-CD11b-) NK cells are the most immature, with no effector ability. R1 (CD27+CD11b-) NK cells have some IFN-γ production capacity, but limited cytotoxicity and are also considered immature. Transitional R2 (CD27+CD11b+) produce high amounts of IFN-γ but mid-levels of GzmB. Finally, mature R3 (CD27-CD11b+) NK cells have the highest levels of GzmB and cytolytic capacity (6,7). In addition, highly mature NK cells also upregulate the expression of the inhibitory receptor KLRG1. Viral infections, including HIV and HCV infections, can have significant effects on the maturation status of NK cells (8–17). For example, at 8 days post infection (dpi) of C57BL/6 (B6) mice with CL13, NK cells become highly activated and mature, with increased frequencies of GzmB+ KLRG1+ R3 NK cells (8–10). In contrast, at 30 dpi with CL13, NK cells become highly immature, with reduced expression of CD11b, and poor responses to new stimuli (8,10). Type I interferon (IFN-I) comprises IFN-α and IFN-β cytokines, which are promptly produced following viral infection. They can interfere with viral replication, initiate, and enhance antigen presentation and broadly prime innate and adaptive immune responses. IFN-I signals trough a heterodimeric IFN-I receptor (IFNAR) comprises two subunits, the IFNAR1 (IFN-α/β receptor α chain) and IFNAR2 (IFN-α/β receptor β chain), which together are expressed in most cells. When IFN-I binds to IFNAR, this leads to its dimerization and downstream signaling, ultimately inducing the expression of hundreds of interferon stimulated genes (ISGs) (18). During CL13 infection, IFN-I production peaks at 0.5-1 dpi (19–21). This early production of IFN-I contributes to viral infection control (20,21). However, IFN-I can also play a detrimental role during the chronic phase of CL13 infection, because it is associated with elevated expression of immunosuppressive IL-10 and PDL-1 in infected DCs and blockade of IFN-I can hasten the clearance of the virus (20,21). Yet, to what extent IFN-I contribute to the effects of viral infection on the maturation of 99 NK cells is not known. Here we aimed to understand how IFN-I modulates NK cells during the acute and chronic phases of CL13 infection. With this purpose, we analyzed the impact of IFNAR1 or IFN-β deficiency on NK cells maturation, activation and effector function at 8 or 30 dpi with CL13 by measuring CD11b, CD27, KLRG1 and GzmB expression. Our results indicate that IFN-I impacts NK cell maturation during CL13 infection and that IFN-α and IFN-β may provide additive maturational cues to NK cells during the acute phase of infection but may have opposing effects during the chronic phase of the infection, with IFN-β promoting and IFN-α restraining maturation. Materials and methods Mice All experiments were approved by the Thomas Jefferson University Institutional Animal Care and Use Committee (IACUC). C57BL/6 (B6, CD45.2) mice breeders were purchased from Charles River. NCI B6-Ly5.1/Cr B6 CD45.1 (B6-CD45.1) mice (Charles River). Ifnar1 -/- mice (CD45.2), which do not express the IFNAR1 chain of the IFNAR receptor, (22,23) backcrossed to the B6 strain were a gift from Dr. Thomas Moran, Mount Sinai School of Medicine, New York, NY (24). C57BL/6Ifnb1tm1(lacZ) mice (herein Ifnb1 -/- mice), which lack IFN-β, were obtained from the Kumamoto University Animal Facilities – Institute of Resource Development and Analysis Center for Animal Resources and Development (25). All mouse strains were bred in house. Male and female mice were 6-10 weeks old when used for experiments. Viruses The CL13 virus strain was a kind gift of Dr. E. John Wherry (University of Pennsylvania, Philadelphia, PA). CL13 was propagated and organ viral loads were quantified as described previously (26). CL13 infections were performed intravenously with 2x106 plaque forming units (pfu). Flow cytometry Cell staining for flow-cytometry was performed as previously described (10). The following antibodies were used: anti-NK 1.1 (clone PK136, APC or BV605, Biolegend), anti-TCR-β (clone H57597, BV605 or BV421 Biolegend; clone H57-597, BV786, BD), anti-CD45.1 (clone A20, PE or PE-Cy7, Biolegend), anti-CD45.2 (clone 104, APC or PercP C5.5, Biolegend) anti-CD11b (clone M1/70, 100 BUV395, BD), anti-CD27 (clone LG.3A10, PerCP-Cy5.5, Biolegend), anti-KLRG1 (clone 2F1/KLRG1, PECy7 or APC, Biolegend), anti-granzyme B (clone GB11, Pacific Blue, Biolegend). Data were acquired with a BD LSRFORTESSA TM cytometer and analyzed with FlowJo TM version 10 (Treestar). Mixed bone marrow chimeras To generated mixed bone marrow chimeras where we could distinguish the cells derived from either of the two donors or from the host, 6-8 weeks old B6-CD45.1 (CD45.1+ CD45.2-) mice were irradiated with 900 Rad and reconstituted with 5x106 bone marrow cells from F1[B6-CD45.1x B6] mice (CD45.1+ CD45.2+) and Ifnar1 -/- (CD45.1CD45.2+) or B6 (CD45.1CD45.2+) mice in a 1:1 ratio to generate CD45.1+ Ifnar1 -/-→ F1[B6CD45.1x B6] or CD45.1+B6→ F1[B6CD45.1x B6] chimeric mice. Mice were rested for two months and given acidified water for the first 20 days. Statistical analysis Data were analyzed using Prism v6. We used a parametric unpaired t-test to study difference between 2 independent groups or One-Way ANOVA test if more than 2 independent groups. For the Post-Hoc analysis, we used the Tukey’s multiple comparisons test. Experiments were repeated at least twice. For all, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Results and discussion At steady state, B6, Ifnar1-/- and Ifnb1 -/- mice had comparable numbers of splenocytes (Figure 1A). At 8 dpi with CL13, the number of splenocytes were similar to those in naïve mice in B6 and Ifnb1- /- mice but were significantly increased in Ifnar1 -/- mice (Figure 1A). Also, compared to B6 mice, the virus titers in the kidneys of Ifnar1-/- mice were slightly but significantly increase (Figure 1B) supporting previous observations that IFN-I signaling helps control CL13 (20,27). On the other hand, Ifnb1 -/- mice did not have increased virus indicating that IFN-α alone can contribute to virus reduction. We and others have shown that B6 mice have reduced frequencies and numbers of NK cells at 8 dpi with CL13, (8,10). Here, we observed a comparable phenotype in infected Ifnar1 -/- and IfnbLacZ mice (Figure 1C), suggesting that the reduction in NK cells is not because of IFN-I. Of note, Ifnar1 -/- mice had higher numbers of NK cells in the spleen compared to Ifnb1-/- mice, which most likely reflect splenocyte number differences between these mice (Figure 1A). 101 Figure 1. IFN-I signaling and NK cells in early chronic CL13 infection A) Number of splenocytes in the indicated mice. B) LCMV titers in the kidneys of the indicated mice at 8 dpi with CL-13 C) Flow cytometry dot-plots representing the gating strategy utilized for the definition of NK cells (TCR-NK 1.1+) (left) with the frequencies and numbers in the spleen (right). D) Flow-cytometry dot-plots with the gating strategy used to define the R1, R2 and R3 NK cell subpopulations, and cumulative frequencies of R1, R and R3 NK cells in the spleens of the indicated mice. Flow-cytometry dot-plots indicating the gating strategy used to define GzmB+ (E) or KLRG1+ (G) NK cells, and cumulative frequencies of GzmB+ or KLRG1+ NK cells in the spleen. All data was collected following 8 dpi with CL13. Graphs display at least 2 similar and independent experiments, with 6-13 mice per group each. In all graphs, data is shown as mean  SEM. 102 Figure 2. IFN-I signaling and NK cells in established chronic CL13 infection A) Number of splenocytes in the indicated mice. B) LCMV titers in the kidneys of the indicated mice at 30 dpi with CL-13. C) NK cell frequencies and calculated total numbers in the spleen of the indicated mice. D) Frequency of R1, R2 and R3 NK cell subpopulations in the spleen of the indicated mice. E) Frequency of NK cells expressing GzmB (left) or KLRG1 (right) in the spleen of the indicated mice. F) Schematic representation of the process involved in creating F1[B6CD45.1x B6] + Ifnar1 -/-→ CD45.1 or F1[B6CD45.1x B6] +B6→ CD45.1 chimeric mice. The gating strategy for each NK cell compartment within a mouse is represented on the right G) CL13 virus titers in the kidneys of the indicated mouse chimeras at 30 dpi. H) Frequency of R1, R2, and R3 NK cell subpopulations, within the B6 or Ifnar1 - /- NK cell compartments in naïve or CL13 infected F1[B6CD45.1x B6] +Ifnar1-/-→ CD45.1 chimeras. I) Frequency of NK cells of B6 or Ifnar1 -/- origin expressing GzmB in the spleens of F1[B6CD45.1x B6] + Ifnar1 -/-→ CD45.1 mice. All data was collected at 30 dpi with CL13. Each graph displays data from at least 2 similar and independent experiments, with 4-10 mice per group and data is shown as mean  SEM. 103 We next assessed the maturation status of NK cells in these mice using CD27 and CD11b as markers. At baseline, naïve B6 and Ifnar1 -/- mice showed comparable frequencies of R1, R2 and R3 NK cells, while naïve Ifnb1 -/- mice showed increased frequencies of R2 NK cells (ANOVA, p<0.05), and decreased frequencies of R3 NK cells (ANOVA, p<0.0001) (Figure 1D). These observations suggest that IFN-I may have a role in the steady state maturation of NK cell and that IFN-β and IFN-α may exert opposing effects in this process. Previously, we and others have shown that compared to naive mice, the NK cells at 8 dpi with CL13 become more mature with increased frequencies of mature R3 at the expense of transitional R2 cells, and also become activated as indicated by increased frequencies of GzmB+ and KLRG1+ NK cells. Notably, we now found that CL13 infection in Ifnar1-/- mice increased the frequencies of immature R1 (p<0.001) while reducing the transitional R2 NK cells (p<0.001). This did not occur in Ifnb1 -/- mice were NK cells became more mature with increased frequencies of R3 cells (p<0.05), but significantly less than in B6 mice (p<0.01). Thus, IFN-I has a critical role in the maturation of NK cells induced by acute viral infection Also, while the frequencies of GzmB+ (Figure 1E) and KLRG1+ (Figure 1G) NK cells increased significantly in Ifnar1 -/- mice, these increases were significantly smaller than in B6 mice indicating that IFN-I plays a role but is not absolutely necessary for the activation of NK cells during acute infection. On the other hand, Ifnb1 -/- mice had a slightly reduced frequency of GzmB+ and normal frequency of KLRG1+ NK cells. Our data thus far indicate that IFN-I signaling is pivotal for optimal NK cell response during th acute phase of CL13 infection. The early peak of IFN-I production following CL13 infection occurs ~1224h post CL13 infection (21,28). As the infection progresses, IFN-I decreases to baseline levels (19). Yet, current evidence suggests there are still remnants of IFN-I activity during the chronic phase of the infection, with increased expression of IFN-I inducible genes (ISGs, including Mx2 , Oas1a , Oas1g , Oas2 , Oas3 , and others (20). Therefore, we next analyzed how IFN-I affects the NK cell phenotype of NK cells during the chronic phase of CL13 infection (30 dpi). We found that CL13 chronically infected B6 and Ifnb1-/- mice had decreased numbers of splenocytes compared to naïve mice (t-test, p<0.05) while Ifnar1 -/- mice had increased numbers of splenocytes when compared to all other groups (Figure 2A). Others have shown that the blockade of IFNAR or IFN-β alone during chronic CL13 infection, results in reduced virus loads in several organs (20,29). In contrast, we found that in Ifnar1 -/- mice and Ifnb1 -/- 104 mice had increased and comparable virus burden (Figure 2B), indicating that IFN-I contribute to virus control in chronic infections. Compared to naïve mice and similar to the acute phase of the infection, the frequencies and absolute numbers of NK cells were reduced in B6, Ifnar1-/- and Ifnb1 -/- (Figure 2C and (10)) with Ifnar1 - /- mice having slightly higher absolute numbers possibly due to the splenomegaly. NK cells from all infected groups displayed an immature phenotype with significantly increased frequencies of R1 at the expense of R2 NK cells, but the frequency or R1 was significantly much higher and the frequency of R2 NK cells was significantly much lower in Ifnb1 -/- mice than in B6 and Ifnar1-/- mice (Figure 2D) This suggests that IFN-β is critical to restrain a shift to an immature phenotype in NK cells during the chronic phase of the infection and that the roles of IFN-α and IFN-β are nonsynergistic during chronic infection. Because the defects were more pronounced in the maturation and activation of NK cells in Ifnb1-/- mice, one would assume that IFN-β signaling becomes more important in activating NK cells as the CL13 infection progresses. Chronic CL13 infection also resulted in increased frequencies of GzmB+ and KLRG1+ NK cells in B6, Ifnar1 -/- and Ifnb1-/- mice, with Ifnb1-/- mice being slightly less efficient at upregulating KLRG1 (Figure 2E). This indicates that the activation of NK cells and their production of GzmB is independent of IFN-I during chronic CL13 infection. These results were unexpected, because IFNAR blockade during chronic CL13 infection results in decreased NK cell cytotoxicity (9). In the experiments in Figure 2E, it was possible that the increase virus titers in the absence of IFN-I signaling resulted in NK cell activation through alternative pathways. Thus, we next tested the role of intrinsic IFN-I signaling for NK cell maturation and activation during the chronic phase of the infection using F1[B6 CD45.1x B6] + Ifnar1 -/-→ CD45.1 and control F1[B6CD45.1x B6] +B6→ CD45.1 chimeric mice (Figure 2F). Both groups had comparable virus loads in their kidneys (Figure 2G) which allowed us evaluating the intrinsic requirement for IFN-I signaling in NK cells maturation and activation independently of virus loads. Both IFNAR-sufficient and IFNAR-deficient NK cells from infected F1[B6CD45.1x B6] + Ifnar1 -/-→ CD45.1 mice had increased frequencies of R1 at the expense of R2 NK cells and R3 NK cells (ANOVA, p<0.0001 for all) (Figure 2H). Also, the WT NK cells from infected F1[B6CD45.1x B6] + Ifnar1 -/-→ CD45.1 had increased frequency of GzmB+ NK cells as compared to similar cells from naïve chimeras while Ifnar1 -/- NK cells did not (Figure 2I). This indicates that IFN-I 111 Abstract Introduction 112 113 Results 114 115 116 117 118 119 120 127 Part III - Discussion 128 CL13 infection renders NK cells unable to promptly respond to unrelated ECTV infection We used chronic CL13 infection and acute Arm infection to understand how chronic viral infection and convalescence from acute infection shapes the NK cell phenotype and response to a subsequent infection with an unrelated virus. Before our work, Whitmire and colleagues had shown that both Arm and CL13 infections lead to reduced numbers of NK cells during the acute phase of the infection (1). Here we show that low NK cell numbers persist in the long-term not only in the case of chronic CL13 infection, but also in mice convalescent from Arm, where virus is no longer detectable by plaque assay as early as 8dpi. This is puzzling and suggests that viral infection with LCMV impacts NK cells in a long-lasting manner independently of chronicity. The numbers of NK cells reach a nadir at 15 dpi with either CL13 or Arm infection, with a lower value in CL13 infected mice. Interestingly, at 30-35 dpi NK cell express Ki67 and their numbers are higher than at 15 dpi, suggesting that they can eventually recover. At 8 dpi with CL13 or ARM, NK cells show a strongly activated and mature phenotype. The frequencies of R3 NK cells dramatically increase, while there are clear signs of strong activation, with high levels of NK cells producing GzmB and expressing KLRG1. Again, the magnitude of this response is higher with Arm than with CL13 infection, particularly regarding the expansion of the R3 NK cell subset. As both infections progress, NK cells develop an immature phenotype, with progressively lower frequencies of R3 NK cells and increased frequencies of the highly immature R1 NK cells, reaching a peak at 15 dpi with Arm. Later, the profile of NK cells begins to improve in Arm infected mice, with increased frequencies of more mature and functional R2 NK cells. In contrast, CL13 infected mice show growing frequencies of R1 NK cells, and limited recovery of R2 NK cell frequencies. Interestingly, the changes in maturation correlated with decreased expression of activating Ly49H and inhibitory Ly49C/I receptors which reach their nadir at 15 dpi with Arm but persist with CL13 at 30-35 dpi. This is surprising because NK cells acquire expression of Ly49 receptors at very early stages of maturation in the bone marrow following interaction with stroma cells (2,3). Altogether, we hypothesize that following Arm and CL13 infection, NK cells respond promptly to infection and then a negative feedback mechanism enters into action: the expression of the inhibitory receptor KLRG1 increases, suppressing NK cells effector activity. After that, terminally differentiated R3 NK cells may die and be replaced by a pool of immature NK cells that migrates from the bone marrow. 129 While this process may be effective after Arm infection, in which there is a NK cell repopulation of more mature cells in the periphery in the absence of virus at 30-35 dpi, during CL13 infection, the persisting viral stimuli does not allow an interruption of the negative feedback loop, resulting in immaturity and reduced numbers of NK cells in the periphery as late as 30-35 dpi. Our data supports this theory to a limited extent. We have found CL13 enters the bone marrow, because we detected viral RNA there (data not shown), even at 30-35 dpi. Hence, virus stimuli is widespread, and wherever we found virus, we also detected reduced numbers of NK cells, and an immature phenotype in those that remain. To fully prove this theory, we could evaluate to what extent CL13 infection in the bone marrow affects the interaction of NK cells with bone marrow stromal cells. It is known that CL13 infection induces persistent structural changes of the stroma of lymphoid organs in the periphery (4,5). It is therefore possible that similar structural changes also occur in the bone marrow. However, Arm infection is not known to induce changes in the stroma of periphery lymphoid organs (4,5). Also, we have previously reported that alteration in the stroma of the bone marrow leads to defective NK cells maturation in old wild type (WT) mice and that leads to the accumulation of immature NK cells in the periphery (6). Microscopy imaging of the bone marrow of CL13 and Arm infected mice, at various timepoints post infection should help address this question. Also, we could evaluate NK cell death, such as by the incorporation of PI and the binding of Annexin V. Furthermore, proliferation assays should be optimized to evaluate incorporation of BrdU at several timepoints post infection and in several organs, including bone marrow and spleen. Finally, we could evaluate migration of NK cells from the bone marrow to the periphery, possibly by optimizing a technique in which cells from a CD45.1 implanted femur are exposed to infection within a CD45.2 host and then assess their migration to the periphery after CL13 infection. Despite their immaturity, NK cells in chronically infected mice have a relatively activated phenotype, with increased expression of GzmB, IFN-γ and KLRG1. A recent report showed similar data regarding persistent activation but did not identify the immature phenotype. Interestingly, they showed that the pre-activated NK cells were more potent at eliminating B16 melanoma tumors than NK cells in naïve mice, indicating that the pre-activation may be beneficial to combat tumors (7). Yet, in the case of ECTV infection, persistent activation was detrimental as NK cells in CL13+ECTV mice failed to become 130 fully activated as demonstrated by the lack of further GzmB, IFN-γ and KLRG1 upregulation and did not protect from mousepox. NK cells have a crucial role in the intrinsic resistance of B6 mice to mousepox (6,8). Here we demonstrated that B6 mice chronically infected with CL13 lose their intrinsic resistance to ECTV, whereas mice that recover from Arm mostly survive. Following 5 dpi with ECTV, NK cells from previously naïve mice or convalescent from acute Arm infection, show extensive proliferation, particularly among the highly functional R2 NK cells, as indicated by increased Ki67 expression and abundant incorporation of administered BrdU. This was particularly notorious in Arm convalescent mice, possibly due to their lower numbers of NK cells. In contrast NK cells from CL13+ECTV mice showed minimal proliferation and expansion. At 5 dpi with ECTV, there were very high frequencies of NK cells in the spleens of ARM+ECTV and Ø+ECTV mice that produced GzmB and IFN-γ. Notably, the negative feedback loop that we observed after LCMV infection was also apparent after ECTV infection. In both Arm+ECTV and Ø+ECTV there was a sharp upregulation of KLRG1, possibly indicating high levels of NK cell activation that need to be suppressed to prevent immunopathology (9,10). In contrast, NK cells from CL13+ECTV infected mice showed impaired GzmB production and minimal increases in IFN-γ production. To test whether this would translate into decreased NK cell cytolytic activity in vivo , we co-transferred tap1-/- and B6 splenocytes into the different mouse groups. Full fledge effectors NK cells, can recognize the lower MHC-I expression of tap1-/- splenocytes compared to WT and preferentially kill them (11,12). We observed that NK cells from naïve, Arm or CL13 infected B6 mice preferentially kill tap1-/- splenocytes. This is quite surprising, since numbers of NK cells are much lower in LCMV infected mice than in naïve mice. However it coincides with recent experiments that showed that NK cells from chronically CL13 infected mice have increased in vitro killing activity (7). Yet, after ECTV infection, which activates NK cells, preferential killing of tap1 -/- cells increased in the Ø+ECTV and in the Arm+ECTV groups but not in CL13+ECTV mice. This was likely due to the inability of their NK cells to become activated and not to the reduced NK cell numbers because Arm recovered mice had reduced NK cell numbers too. In summary, our data indicate that despite increased basal activation levels, chronic infection can result in impaired NK cell maturation and activation in response to secondary infections, which can contribute to increased susceptibility to opportunistic infections in chronically infected individuals. 131 The recruitment of NK cells to respond to ECTV infection in chronically CL13 infected mice is likely reduced CXCL9 is an important chemokine for the recruitment of NK cells (13). Following ECTV infection, DCs and monocytes produce large amounts of CXCL9 that is recognized by immature NK cells via the CXCR3 receptor (13–15). This leads to the recruitment of fully fledged cytolytic R3 NK cells, critical in the control of ECTV infection (8,9). Simultaneously in this process, they downregulate the expression of the CXCR3 receptor (16). One may hypothesize that NK cells may develop some level of tissue residency following infection, and therefore should not be recruited elsewhere. This was already described to a certain degree to occur during LCMV infection, with the persistence of a population of liver-resident NK cells (17). Interestingly, we see that NK cells downregulate the expression of the CXCR3 receptor in Ø+ECTV and Arm+ECTV infected WT mice but fail to do so in CL13+ECTV infected mice. This further strengthens our hypothesis in which CL13 renders NK cells arrested in an immature state. Yet, it also supports the hypothesis that the process leading to the recruitment of NK cells in CL13+ECTV infected mice is impaired. In fact, we observed that the numbers of monocytes and DCs, powerful producers of the recruiting chemokine CXCL9, did not increased in Ø+ECTV and Arm+ECTV but not in CL13+ECTV mice (data not shown). Moreover, the frequency of both DCs and monocytes producing CXCL9 was much lower in CL13+ECTV infected mice than in Ø+ECTV or Arm+ECTV, where CXCL9 production increased substantially following ECTV infection (data not shown). 132 IFN-I signaling is required for normal NK cell development during CL13 infection There is plenty of research about the role of IFN-I signaling in the establishment of chronic CL13 infection. IFN-I production peaks shortly after CL13 infection, yet progressively decreases from there to values that may be lower than in previously naïve mice (18). Despite this fact, many associate the presence of IFN-I signaling to the persistence of chronic CL13 infection (19,20). Others consider IFN-I to be pivotal in eliciting effective anti-viral responses (21). It was previously shown that IFN-I signaling is important for NK cell maturation after viral infection (22). Moreover, NK cells have been associated for a long time with the establishment of chronic CL13 infection (23–25). Therefore, one of the hypothesis currently defended is that NKG2Dmediated activation of NK cells results in the killing of CD4+ T cells (23–25). With this in mind, we hypothesized that IFN-I signaling could lead to the expansion of dysfunctional NK cells. To test for this, we infected with CL13 Ifnar1 -/- and Ifnb-/- mice, in which both IFNα and IFN-β, or only IFN-β signaling is absent and observed the NK cell phenotype at different timepoints. We found that both mouse strains had increased virus loads at 8 dpi, suggesting this IFN-I production is critical to the early control of CL13 infection. Interestingly, both strains showed impaired NK cell maturation, activation and effector activity. At 8 dpi with CL13, NK cells of Ifnar1 -/- mice had higher frequencies of immature R1 NK cells, minimal frequencies of differentiated R3 NK cells, and reduced frequencies of GzmB+ and KLRG1+ cells as compare with WT mice with Ifnb-/- mice having an intermediate phenotype . This suggests that both IFN-α and IFN-β contribute to the proper maturation and activation of NK cell at 8 dpi with CL13. Our data also suggests that IFN-I signaling is required for the long term control of CL13 because both, Ifnar1 -/- and Ifnb -/- mice had increased CL13 virus loads at 30 dpi and reduced numbers of NK cells. Interestingly, maturation of NK cells in infected Ifnar1 -/- and WT mice was similarly impaired, with higher frequencies of immature R1 NK cells and lower frequencies of functional R2 NK cells as compared to naïve mice. Interestingly, Ifnb-/- mice had more severe maturation defects and lower frequencies of GzmB+ NK cells suggesting that as CL13 infection progresses, IFN-β becomes increasingly pivotal in preserving some of the NK cell function. 133 We also showed that IFN-I signaling is important for NK cells to preserve effector function at 30 dpi with CL13. In B6+ Ifnar1 -/-→B6 bone marrow chimeras NK cells have comparable maturation defects in the B6 and in the Ifnar1 -/- compartments at 30 dpi with CL13. Yet, production of GzmB was higher in the B6 than in the Ifnar1 -/- NK cell populations. We could complement our work in the role for IFN-I signaling for NK cell maturation, activation and function with several experiments. First, we could evaluate what happens with NK cells from the bone marrow chimeras above at 8 dpi with CL13. Also, we could determine how IFN-α and IFN-β differ in their roles in shaping NK cells. Unfortunately, there is not yet an IFN-α-/- mouse strain, which complicates our assessments. Alternatively, we could design new experiments in which either IFN-α or IFN-β are blocked at different timepoints of CL13 infection, and then assess how that changes the NK cell profile and their ability to kill protective CD4+ T cells. 134 ECTV-specific T-cell maturation and activation are impaired during CL13 infection, but response is possible following priming with attenuated ECTV-Δ36 CL13 induces strong exhaustion of T cells, yielding them unresponsive to infection, with decreased polyfunctional activity and deletion of many of the LCMV specific T-cell clones (26–30). We now reveal a previously unknown facet in which the CD8+ T cells response towards an unrelated pathogen is impaired. At 7 dpi with ECTV, we observed the expansion of T cells in Ø+ECTV and Arm+ECTV but not in CL13+ECTV (31–33). We also showed that following ECTV infection, T cells in CL13+ECTV infected mice proliferate less, with reduced expression of Ki67 and reduced incorporation of BrdU, than in Ø+ECTV and Arm+ECTV infected mice. Yet, some of the effects of LCMV infection should persist following clearance of infection, because expression of Ki67 in Arm+ECTV infected mice was lower than in Ø+ECTV. Notably, when we adoptively transferred total splenocytes from naïve WT mice, theoretically sufficient to curb ECTV infection (32), the transferred T cells expanded less in CL13+ECTV than in Ø+ECTV. This occurred despite that the CL13+ECTV had higher antigen load than Ø+ECTV infected group. Similar results were obtained after transfer of OVA-specific cells OT-I CD8 T cells and challenge with ECTV-OVA. This data suggests that cell-extrinsic contribute to the impaired T cell expansion in CL13+ECTV mice. We also found that the activation of T cells following ECTV infection was significantly diminished in CL13+ECTV mice. As showed above, CD8+ T cells in CL13 infected mice had increased frequency of T cells expressing the maturation marker CD44 and decreased frequency of cells expressing the lymph node homing marker CD62L (28). Yet, after ECTV infection, the frequency of GzmB+ CD8+ T cells in CL13+ECTV increased to much lower levels than in Ø+ECTV or Arm+ECTV mice. We hypothesize that the unresponsiveness to ECTV infection was due to the suppressive effects of inhibitory receptors. As described before, we showed that CD8+ T cells in CL13 infected mice had increased frequencies of cells expressing the inhibitory receptors KLRG1 and PD1 (34–38). Since blockade of inhibitory receptors is associated with rescue of T-cell function during CL13 infection (35–40), we blocked PD1 signaling with anti-PD1 monoclonal antibodies, either alone or together with anti-TIGIT monoclonal antibodies and we saw minimal improvement effects in ECTV virus control (data not shown). 135 The expansion of CD8+ T cells specific for the immunodominant ECTV epitope TSYKFESV was also reduced in CL13+ECTV as compared to Ø+ECTV and Arm+ECTV mice. Notably, the few TSYKFESV specific CD8+ T cells that did expand in CL13+ECTV also had defective activation, with decreased production of GzmB, increased expression of CD62L and decreased expression of KLRG1. Since these are de novo differentiated CD8+ T cells, we hypothesized that circulatory factors should be suppressing the development of effector activity in CD8+ T cells. IL-10 has been abundantly described has pivotal in the establishment of chronicity during CL13 infection; its levels remain increased throughout CL13 chronic infection, and it is associated with decreased TCR signal transduction (19,41–45). To bypass the effects of IL-10 mediated immunosuppression, we adoptively transferred IL10R -/- total splenocytes to CL13+ECTV infected mice. This provided some increase in time-to-death but not in survival time compared to CL13+ECTV receiving WT splenocytes. This suggests that various mechanisms contribute to the T-cell unresponsiveness to unrelated pathogens in CL13 infected mice. There is abundant literature suggesting that persistent IFN-I leads to T cell unresponsiveness during CL13 infection (19,20). Yet, Ifnar -/- splenocytes adoptively transferred into CL13+ECTV mice had limited effects on virus control and did not expand TSYKFESV-specific CD8+ T cells at 7 dpi with ECTV (data not shown). In contrast, following ECTV Δ166 infection, which is severely attenuated because it lacks an IFN-I decoy receptor (46), 50% of mice previously infected with CL13 survived and mounted TSYKFESV specific CD8+ T cell responses that were as strong as those in previously naïve mice. This suggests that optimizing IFN-I signaling may help to promoted ECTV virus control in the setting of chronic CL13 infection. Since CL13+ECTV mice mounted limited but clear TSYKFESV-specific CD8+ T cell responses, we hypothesized that if they had more time to respond, they would be able to effectively respond and protect mice from ECTV infection. To test this, we infected mice with the highly attenuated ECTV virus strain ECTV-Δ36 (47). and found that the number of TSYKFESV-specific CD8+ T cells in Ø+ECTV-Δ36 and CL13+ECTV-Δ36 were similar at 7, 15 and 30 dpi with ECTV-Δ36. At this later timepoint, and in contrast to 7 and 15 dpi, there was also an effective antibody response to ECTV-Δ36. 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