Direct Presentation Is Sufficient for an Efficient Anti-Viral CD8+ T Cell Response

The extent to which direct- and cross-presentation (DP and CP) contribute to the priming of CD8+ T cell (TCD8+) responses to viruses is unclear mainly because of the difficulty in separating the two processes. Hence, while CP in the absence of DP has been clearly demonstrated, induction of an anti-viral TCD8+ response that excludes CP has never been purposely shown. Using vaccinia virus (VACV), which has been used as the vaccine to rid the world of smallpox and is proposed as a vector for many other vaccines, we show that DP is the main mechanism for the priming of an anti-viral TCD8+ response. These findings provide important insights to our understanding of how one of the most effective anti-viral vaccines induces immunity and should contribute to the development of novel vaccines

Mechanisms of memory CD8+ T cell protection from an acuute lethal viral disease

CD8+ T cells are important mediators of adaptive immunity that work to eliminate certain pathogenic viruses. Antigen-specific CD8 + T cells exert their antiviral effects via direct cytolysis of infected cells and through the production of cytokines and chemokines. The experiments described in this dissertation identify the essential components in memory CD8+ T cell mediated protection against an acute lethal viral infection. The two major anti-viral effector mechanisms of CD8+ T cells are thought to be perforin (Prf)-mediated cell lysis and interferon gamma (IFN-γ)-mediated induction of an anti-viral state. By affecting the expression of proteins involved in antigen presentation, IFN-γ is also thought to shape the magnitude and specificity of the CD8+ T cell response. Therefore, in our first manuscript, we studied the role of Prf and IFN-γ in shaping the effector and memory CD8+ T cell response to vaccinia virus (VACV). We found that the virus loads are the main reason for the increased strength of the CD8 response in IFN-γ and Prf deficient mice and that neither Prf nor IFN-γ deficiency had an effect on the immunodominance hierarchy of 5 Kb-restricted CD8+ T cell determinants during either acute infection or after recovery. Thus, our work demonstrated that neither IFN-γ nor Prf are essential for the control of VACV. Moreover, we showed that CD8+ T cell immunodominance during VACV infection was unaffected by the effects of IFN-γ on the antigen presentation machinery. Memory CD8+ T cells induced by immunization with VACV, the virus in the smallpox vaccine, protect from mousepox, the mouse homolog of human smallpox. In our second manuscript, we showed that the ability of memory CD8+ T cells to protect from mousepox depended on their initial frequency and required the presence of IFN-γ. However, while memory CD8+ T cells could provide all the IFN-γ necessary for protection, their ability to produce IFN-γ was dispensable. Thus, the capacity of memory CD8+ T cells to protect from a viral infection was dependent on their frequency but independent of their ability to produce IFN-γ. To follow-up on these findings, in our third manuscript we demonstrated that the protective ability of memory CD8+ T cells was dependent on the secretory perforin-dependent granule exocytosis pathway. Hence, the effector to target cell ratio is greater when the initial memory CD8+ T cell frequency is high. This results in more infected cells being killed by the Prf/granzyme pathway and the survival of the host. We have previously shown that the adoptive transfer of multispecific memory CD8+ T cells can protect from an acute lethal viral disease. Therefore, in our fourth manuscript we determined whether CD8 + T cells specific for single ID or SD peptides could be protective. We found that complete protection from mousepox could be achieved by memory CD8+ T cells specific for single ID or SD determinants induced by immunization with peptide-pulsed dendritic cells. Our data suggests that it is feasible to produce effective anti-viral CD8+ T cell vaccines using single CD8+ T cell determinants. Additionally, we showed that the protective ability of monospecific memory CD8+ T cells was not compromised in the absence of natural killer cells, which are essential for resistance to mousepox in non-immune mice. Together, the work presented in this dissertation contribute to the efforts of rational vaccine development by providing information about mechanisms of acquired protection that may be applicable to other pathogenic viruses that cause acute or chronic viral diseases

Memory CD8 + T cells specific for a single immunodominant or subdominant determinant induced by peptide-dendritic cell immunization protect from an acute lethal viral disease

The antigens recognized by individual CD8 + T cells are small peptides bound to major histocompatibility complex (MHC) class I molecules. The CD8 + T cell response to a virus is restricted to several peptides, and the magnitudes of the effector as well as memory phases of the response to the individual peptides are generally hierarchical. The peptide eliciting a stronger response is called immunodominant (ID), and those with smaller-magnitude responses are termed subdominant (SD). The relative importance of ID and SD determinants in protective immunity remains to be fully elucidated. We previously showed that multispecific memory CD8 + T cells can protect susceptible mice from mousepox, an acute lethal viral disease. It remained unknown, however, whether CD8 + T cells specific for single ID or SD peptides could be protective. Here, we demonstrate that immunization with dendritic cells pulsed with ID and some but not all SD peptides induces memory CD8 + T cells that are fully capable of protecting susceptible mice from mousepox. Additionally, while natural killer (NK) cells are essential for the natural resistance of nonimmune C57BL/6 (B6) to mousepox, we show that memory CD8 + T cells of single specificity also protect B6 mice depleted of NK cells. This suggests it is feasible to produce effective antiviral CD8 + T cell vaccines using single CD8 + T cell determinants and that NK cells are no longer essential when memory CD8 + T cells are present. © 2012, American Society for Microbiology.Peer Reviewe

Memory CD8+ T cells can outsource IFN-γ production but not cytolytic killing for antiviral protection

Immunization with vaccinia virus (VACV), the virus comprising the smallpox vaccine, induces memory CD8+ T cells that protect from subsequent infections with smallpox in humans or the related ectromelia virus (ECTV) in mice. Memory CD8+ T cells largely mediate these effects by expanding into secondary effectors that secrete the antiviral cytokine interferon-γ (IFN-γ) and induce cytolysis via releasing factors such as perforin, which permeabilizes target cells. We show that protection from ECTV infection after VACV immunization depends on the initial memory cell frequency and ability of expanded secondary effectors to kill infected targets in a perforin-dependent manner. Although IFN-γ is essential for antiviral protection, it can be produced by either secondary effectors or concomitant primary effector CD8 + T cells recruited to the response. Thus, during lethal virus challenge, memory CD8+ T cells are required for cytolytic killing of infected cells, but primary effectors can play important roles by producing IFN-γ. © 2013 Elsevier Inc.R01AI048849, R01AI065544, 5U19AI083008; P30CA006927; NIH T32 CA-009035036Peer Reviewe

Sequential activation of two pathogen-sensing pathways required for type I interferon expression and resistance to an acute DNA virus infection

Toll-like receptor 9 (TLR9), its adaptor MyD88, the downstream transcription factor interferon regulatory factor 7 (IRF7), and type I interferons (IFN-I) are all required for resistance to infection with ectromelia virus (ECTV). However, it is not known how or in which cells these effectors function to promote survival. Here, we showed that after infection with ECTV, the TLR9-MyD88-IRF7 pathway was necessary in CD11c(+) cells for the expression of proinflammatory cytokines and the recruitment of inflammatory monocytes (iMos) to the draining lymph node (dLN). In the dLN, the major producers of IFN-I were infected iMos, which used the DNA sensor-adaptor STING to activate IRF7 and nuclear factor κB (NF-κB) signaling to induce the expression of IFN-α and IFN-β, respectively. Thus, in vivo, two pathways of DNA pathogen sensing act sequentially in two distinct cell types to orchestrate resistance to a viral disease

Crosstalk between the type 1 interferon and nuclear factor kappa B pathways confers resistance to a lethal virus infection

Nuclear factor kappa B (NF-κB) and type 1 interferon (T1-IFN) signaling are innate immune mechanisms activated upon viral infection. However, the role of NF-κB and its interplay with T1-IFN in antiviral immunity is poorly understood. We show that NF-κB is essential for resistance to ectromelia virus (ECTV), a mouse orthopoxvirus related to the virus causing human smallpox. Additionally, an ECTV mutant lacking an NF-κB inhibitor activates NF-κB more effectively in vivo, resulting in increased proinflammatory molecule transcription in uninfected cells and organs and decreased viral replication. Unexpectedly, NF-κB activation compensates for genetic defects in the T1-IFN pathway, such as a deficiency in the IRF7 transcription factor, resulting in virus control. Thus, overlap between the T1-IFN and NF-κB pathways allows the host to overcome genetic or pathogen-induced deficiencies in T1-IFN and survive an otherwise lethal poxvirus infection. These findings may also explain why some pathogens target both pathways to cause disease. © 2013 Elsevier Inc.NIAID (U19AI083008 and R01AI065544); NCI (CA006927); Instituto de Salud Carlos III, Spanish Ministry of Health; Spanish Ministry of Science and InnovationPeer Reviewe