Human cytomegalovirus (HCMV) is a paradigm for viral immune evasion strategies yet, paradoxically, primary infection of immunocompetent individuals rarely causes serious disease and such primary infections are normally quickly resolved. In contrast, infection of individuals whose immune systems are compromised (such as HIV/AIDS patients and transplant patients) or immature (such as the foetus in utero) often leads to wide spread viral replication and dissemination and this can often be life threatening. Although the immune evasion mechanisms employed by HCMV in vitro are well documented, the effectiveness of these during primary infection in vivo is not absolutely clear but probably allow the pathogen to initially overcome primary immune responses and thus create a window of opportunity for the virus to replicate and disseminate to cell types efficiently enough for latency to be established. Such a strategy could be conceived to be optimal for a life-long persistent pathogen as unchecked viral replication, leading to host mortality, would clearly be a dead end strategy for any virus. However, the establishment of quiescence would also, in itself, be a biological dead end for the virus, unless it was able to reactivate and re-establish lytic infection in order to infect naïve individuals. Similarly, a fitting time for a comprehensive set of immunevasion functions to be employed by the virus would be during reactivation from latency; these would again create a window of opportunity for the virus to re-establish the production of new virions in the face of an existing and primed anti-viral immune response.
Our programme of research seeks to addressed a number of important questions concerning the immunobiology of HCMV both in lytic and during latent infection. With a particular focus on the generation, maintanance and funtion of memory CD4+ and CD8+ T cells and the immune evasion mechanism employed by the virus to modulate Natural Killer cell function.
A key feature of HCMV persistence, in the face of this normally robust host immune response, is the establishment of viral latency. In contrast to lytic infection, which is characterised by extensive viral gene expression and virus production, long-term latency in cells of the myeloid lineage is characterised by highly restricted expression of viral genes, including UL138 and LUNA. Here we report that both UL138 and LUNA-specific T cells were detectable directly ex vivo in healthy HCMV seropositive subjects and that this response is principally CD4+ T cell mediated. These UL138-specific CD4+ T cells are able to mediate MHC class II restricted cytotoxicity and, importantly, show IFNγ effector function in the context of both lytic and latent infection. Furthermore, in contrast to CD4+ T cells specific to antigens expressed solely during lytic infection, both the UL138 and LUNA-specific CD4+ T cell responses included CD4+ T cells that secreted the immunosuppressive cytokine cIL-10. We also show that cIL-10 expressing CD4+ T-cells are directed against latently expressed US28 and UL111A. Taken together, our data show that latency-associated gene products of HCMV generate CD4+ T cell responses in vivo, which are able to elicit effector function in response to both lytic and latently infected cells. Importantly and in contrast to CD4+ T cell populations, which recognise antigens solely expressed during lytic infection, include a subset of cells that secrete the immunosuppressive cytokine cIL-10. This suggests that HCMV skews the T cell responses to latency-associated antigens to one that is overall suppressive in order to sustain latent carriage in vivo.
CD8+ T cells specific for HCMV proteins pp65, IE1 and IE2 are present at high frequencies in HCMV seropositive individuals and these have been shown to have phenotypes associated with terminal differentiation, as well as both cytokine and proliferative dysfunctions, especially in the elderly. However, more recently, T cell responses to many other HCMV proteins have been described but little is known about their phenotype and function. Consequently, in this study, we chose to determine the diversity of HCMV specific CD8+ T cell responses to eleven HCMV ORFs in a cohort of donors aged 20 – 80 years old as well as their ability to secrete IFNγ. Finally, we also tested their functional anti-viral capacity using a novel viral dissemination assay. We identified substantial CD8+ T cell responses by IFNγ ELISPOT assays to all eleven of these HCMV proteins and, across the cohort, individuals displayed a range of responses from the tightly focused to highly diverse which were stable over time. CD8+ T cell responses to the HCMV ORFs were highly differentiated and predominantly CD45RA+, CD57+ and CD28-, across the cohort. These highly differentiated cells had the ability to inhibit viral spread even following direct ex-vivo isolation. Taken together, our data argue that HCMV specific CD8+ T cells have effective anti-viral activity irrespective of the viral protein recognized across the whole cohort and despite viral immune evasion.