NK cells are innate lymphocytes that are crucial for host immunity during infections. Discrimination between healthy and infected cells is facilitated through a sum of inhibitory and stimulatory signals. Host cells can modulate the expression of NK ligands in response to infection, transformation, and stress, while viruses can exploit these mechanisms to prevent the killing of the infected cells. This thesis focuses on the interaction between the NK receptor, NKR-P1B, and its ligand, Clr-b. First, we used a reporter cell-based protocol to establish hybridomas producing monoclonal antibodies against a putative viral immunoevasin, RCTL. We express CD3ΞΆ-fusion proteins on the reporter cells, which we then use to immunize and screen hybridoma specificities. Our results demonstrate a rapid, efficient, and high-throughput method of monoclonal antibody screening, and provide the framework for our work on cytomegalovirus evasion of the NKR-P1B:Clr-b system.
Next, we show that RCMV infection results in a notable downregulation of rClr-b at the protein and transcript levels. Conversely, RCTL is upregulated during infection, and binds to the same NK-inhibitory receptor as Clr-b, NKR-P1B. In the absence of RCTL, RCMV-mediated Clr-b loss leads to increased NK-killing of infected targets and an NK-dependent reduction of viral titers in vivo. Notably, NKR-P1B is highly polymorphic, and certain rat NKR-P1B alleles have lost binding to the viral RCTL but not to the host Clr-b molecule, suggesting co-evolution between the host and the virus.
In the next chapter, we address some of the mechanisms responsible for CMV-mediated Clr-b downregulation, and show that Clr-b downregulation also occurs to mice in response to MCMV infection. Moreover, early gene expression (host or viral) appears to be required for Clr-b downregulation. Interestingly, engagement of any one conventional pattern recognition receptor is insufficient to mimic MCMV-mediated Clr-b downregulation. Similarly, fibroblasts lacking various intermediates for the interferon or inflammasome pathways still downregulate mClr-b, with the exception of the DNA sensor, Zbp-1. Lastly, a recently identified autocatalytic motif conserved in the rat and mouse Clr-b transcripts, the hammerhead ribozyme, appears to be involved in Clr-b regulation.
Taken together, our results explore a novel and important role for NKR-P1B:Clr-b interactions in viral immunity.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/32079 |
| Date | 19 January 2012 |
| Creators | Mesci, Aruz |
| Contributors | Carlyle, James Robert |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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