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The role of natural killer T cells in the early stages of the herpes simplex virus infection in skin

Final Report Summary - NKT IN HERPES (The role of natural killer T cells in the early stages of the herpes simplex virus infection in skin)

Objectives
The overall aim of this study was to define the role of human natural killer T (NKT) cells and their ability to interact with skin-resident innate immune cells during the early stages of herpes simplex virus (HSV) infection. Genital herpes, caused by HSV-1 and -2, has a worldwide prevalence and is the leading cause of genital ulcers. An increasing number of clinical and epidemiological studies indicate that a recent genital HSV-2 infection increases the risk of HIV acquisition and transmission by up to three-fold. Therefore, further studies that examine the local immune responses to HSV-2 infection are necessary to elucidate the mechanisms of enhanced risk of HIV-1 acquisition and reveal possible strategies for the development of an effective HSV vaccine. Our project focused in particular on the responses of human NKT cells, which are poised to respond within hours of activation and can rapidly modulate cellular immune responses.

The specific objectives of the project were:
1. to determine the presence of NKT cells in normal human skin and recurrent herpes lesions and their localisation in relation to other immune stimulatory cells;
2. to establish the nature of CD1d-restricted NKT cell interactions with skin-derived innate immune cells following HSV infection;
3. To study the ability of human NKT cells to directly respond to virus infection through triggering of Toll-like receptors (TLRs) and in concert with the skin-resident immune cells.

Results
NKT cells are activated by self and foreign glycolipids presented in complex with CD1d molecules. Skin-resident keratinocytes express CD1d and are the main targets of HSV infection. Thus, we were interested to examine the interactions between these innate immune cell types during the early stages of HSV infection.

In our study we showed that productive HSV-1 infection in keratinocytes severely inhibited NKT cell activation. Interestingly, CD1d downregulation was not observed on HSV-1-infected keratinocytes as was previously reported for other antigen-presenting cells. In addition, we found that cell-to-cell contact between infected keratinocytes and NKT cells was required for the inhibition, as the cell-free-virus did not affect NKT cell activation. On the other hand, productive infection of NKT cells was not required for the inhibitory effect. Following co-culture with infected cells, NKT cells were no longer responsive to further stimulation with alpha-GalCer-loaded CD1d. We found that exposure of NKT cells to HSV-1-infected cells resulted in impaired TCR signalling downstream of ZAP70. In addition, infected cells were found to upregulate the expression of the negative T cell regulator, galectin-9; however, blocking experiments indicated that the impairment of NKT cell responses was independent of galectin-9.

Our study also showed that despite expression of all known human TLRs, NKT cells alone did not secrete IFN-gamma in direct response to any of the tested TLR ligands. Instead, it appears that NKT cells respond to signals generated from TLR-pre-treated antigen-presenting cells such as dendritic cells.

Conclusions
The major finding of our study is the demonstration of a novel immune evasion strategy utilised by HSV to directly inhibit NKT cell activation. As NKT cells represent one of the earliest sources of IFN-gamma in an infection such immunoevasive measures can be expected to lead to delayed immune activation of other effector cell types and consequently lead to more extensive spread of virus from the initial site of replication in the skin and mucosa. Our results also suggest that the inhibitory effects of HSV infection on NKT cell activation should be taken into account for the design of a more effective genital herpes vaccine.