Mark R. Wills

Functional Heterogeneity and High Frequencies of Cytomegalovirus-Specific CD8+ T Lymphocytes in Healthy Seropositive Donors

ABSTRACT Human cytomegalovirus (HCMV) infection is largely asymptomatic in the immunocompetent host, but remains a major cause of morbidity in immunosuppressed individuals. Using the recently described technique of staining antigen-specific CD8+ T cells with peptide-HLA tetrameric complexes, we have demonstrated high levels of antigen-specific cells specific for HCMV peptides and show that this may exceed 4% of CD8+ T cells in immunocompetent donors. Moreover, by staining with tetramers in combination with antibodies to cell surface markers and intracellular cytokines, we demonstrate functional heterogeneity of HCMV-specific populations. A substantial proportion of these are effector cytotoxic T lymphocytes, as demonstrated by their ability to lyse peptide-pulsed targets in “fresh” killing assays. These data suggest that the immune response to HCMV is periodically boosted by a low level of HCMV replication and that sustained immunological surveillance contributes to the maintenance of host-pathogen homeostasis. These observations should improve our understanding of the immunobiology of persistent viral infection.

Identification of Naive or Antigen-Experienced Human CD8 + T Cells by Expression of Costimulation and Chemokine Receptors: Analysis of the Human Cytomegalovirus-Specific CD8 + T Cell Response

Human CMV (HCMV) infection provides an informative model of how long term human CD8+ T cell memory is maintained in the presence of Ag. To clarify the phenotypic identity of Ag-experienced human CD8+ T cells in vivo, we determined the expression of costimulation and chemokine receptors on Ag-specific CD8+ T cells by quantifying individual virus-specific clones in different cell populations using TCR clonotypic probing. In healthy HCMV carriers, expanded CD8+ clones specific for either HCMV tegument protein pp65 or immediate-early protein IE72 are found in both CD45ROhigh cells and the subpopulation of CD45RAhigh cells that lack the costimulatory molecule CD28. In contrast to previous suggested models of CD8+ T cell memory, we found that in healthy virus carriers highly purified CD28−CD45RAhighCCR7− cells are not terminally differentiated, because following stimulation in vitro with specific HCMV peptide these cells underwent sustained clonal proliferation, up-regulated CD45RO and CCR5, and showed strong peptide-specific cytotoxic activity. In an individual with acute primary HCMV infection, HCMV pp65-specific CD8+ T cells are predominantly CD28−CD45ROhighCCR7−. During convalescence, an increasing proportion of pp65-specific CD8+ T cells were CD28−CD45RAhighCCR7−. We conclude that naive human CD8+ T cells are CD28+CD45RAhigh, express CCR7 but not CCR6, and are predominantly CD27+ and L-selectin CD62 ligand-positive. The phenotype CD27+CD45RAhigh should not be used to identify naive human CD8+ T cells, because CD27+CD45RAhigh cells also contain a significant subpopulation of CD28−CD27+ Ag-experienced expanded clones. Thus CD8+ T cell memory to HCMV is maintained by cells of expanded HCMV-specific clones that show heterogeneity of activation state and costimulation molecular expression within both CD45ROhigh and CD28−CD45RAhigh T cell pools.

Human cytomegalovirus immunity and immune evasion

Human cytomegalovirus (HCMV) infection induces both innate immune responses including Natural Killer cells as well as adaptive humoral and cell mediated (CD4+ helper, CD8+ cytotoxic and γδ T cell) responses which lead to the resolution of acute primary infection. Despite such a robust primary immune response, HCMV is still able to establish latency. Long term memory T cell responses are maintained at high frequency and are thought to prevent clinical disease following periodic reactivation of the virus. As such, a balance is established between the immune response and viral reactivation. Loss of this balance in the immunocompromised host can lead to unchecked viral replication following reactivation of latent virus, with consequent disease and mortality. HCMV encodes multiple immune evasion mechanisms that target both the innate and acquired immune system. This article describes the current understanding of Natural killer cell, antibody and T cell mediated immune responses and the mechanisms that the virus utilizes to subvert these responses.

Human cytomegalovirus encodes an MHC class I-like molecule (UL142) that functions to inhibit NK cell lysis.

Clinical and low passage strains of human CMV (HCMV) encode an additional MHC class I-related molecule UL142, in addition to the previously described UL18. The UL142 open reading frame is encoded within the ULb′ region which is missing from a number of common high passage laboratory strains. Cells expressing UL142 following transfection, and fibroblasts infected with a recombinant adenovirus-expressing UL142, were used to screen both polyclonal NK cells and NK cell clones, in a completely autologous system. Analysis of 100 NK cell clones derived from five donors, revealed 23 clones that were inhibited by fibroblasts expressing UL142 alone. Small-interfering RNA-mediated knockdown of UL142 mRNA expression in HCMV-infected cells resulted in increased sensitivity to lysis. From these data we conclude that UL142 is a novel HCMV-encoded MHC class I-related molecule which inhibits NK cell killing in a clonally dependent manner.

Large clonal expansions of human virus‐specific memory cytotoxic T lymphocytes within the CD57+ CD28– CD8+ T‐cell population

The proportion of human peripheral blood CD8+ T cells that are CD57+ CD28– is low at birth but increases with age and in individuals infected with human cytomegalovirus (HCMV) or human immunodeficiency virus (HIV). These CD57+ CD28– CD8+ T cells contain large oligoclonal T‐cell expansions whose antigen specificity is unknown. We identified clonal expansions of virus‐specific memory cytotoxic T‐lymphocyte precursors (CTLp) in both healthy carriers of HCMV and in asymptomatic HIV‐infected subjects. In each subject, from the T‐cell receptor (TCR) β‐chain hypervariable sequence of each immunodominant CTL clone, we designed complementary oligonucleotide probes to quantify the size and phenotypic segregation of individual virus‐specific CTL clones in highly purified populations of peripheral blood CD8+ T cells. We found large clonal expansions of virus‐specific CTL clonotypes in CD57+ CD28– CD8+ T cells. Using limiting dilution analysis, we found functional peptide‐specific CTLp at high frequency in CD57+ CD28– cells. Thus, memory CTL specific for persistent viruses account for many oligoclonal expansions within CD57+ CD28– CD8+ T cells.

CMV and Immunosenescence: from basics to clinics

Alone among herpesviruses, persistent Cytomegalovirus (CMV) markedly alters the numbers and proportions of peripheral immune cells in infected-vs-uninfected people. Because the rate of CMV infection increases with age in most countries, it has been suggested that it drives or at least exacerbates “immunosenescence”. This contention remains controversial and was the primary subject of the Third International Workshop on CMV & Immunosenescence which was held in Cordoba, Spain, 15-16th March, 2012. Discussions focused on several main themes including the effects of CMV on adaptive immunity and immunosenescence, characterization of CMV-specific T cells, impact of CMV infection and ageing on innate immunity, and finally, most important, the clinical implications of immunosenescence and CMV infection. Here we summarize the major findings of this workshop.

Dynamics of T cell memory in human cytomegalovirus infection

Primary human cytomegalovirus (HCMV) infection of an immunocompetent individual leads to the generation of a robust CD4+ and CD8+ T cell response which subsequently controls viral replication. HCMV is never cleared from the host and enters into latency with periodic reactivation and viral replication, which is controlled by reactivation of the memory T cells. In this article, we discuss the magnitude, phenotype and clonality of the T cell response following primary HCMV infection, the selection of responding T cells into the long-term memory pool and maintenance of this memory T cell population in the face of a latent/persistent infection. The article also considers the effect that this long-term surveillance of HCMV has on the T cell memory phenotype, their differentiation, function and the associated concepts of T cell memory inflation and immunosenescence.

NKG2D Ligand MICA Is Retained in the cis-Golgi Apparatus by Human Cytomegalovirus Protein UL142

ABSTRACT Human cytomegalovirus (HCMV) evades T-cell recognition by down-regulating expression of major histocompatibility complex (MHC) class I and II molecules on the surfaces of infected cells. Contrary to the “missing-self” hypothesis, HCMV-infected cells are refractory to lysis by natural killer (NK) cells. Inhibition of NK cell function is mediated by a number of HCMV immune evasion molecules, which operate by delivering inhibitory signals to NK cells and preventing engagement of activating ligands. One such molecule is UL142, which is an MHC class I-related glycoprotein encoded by clinical isolates and low-passage-number strains of HCMV. UL142 is known to down-modulate surface expression of MHC class I-related chain A (MICA), which is a ligand of the activating NK receptor NKG2D. However, the mechanism by which UL142 interferes with MICA is unknown. Here, we show that UL142 localizes predominantly to the endoplasmic reticulum (ER) and cis-Golgi apparatus. The transmembrane domain of UL142 mediates its ER localization, while we propose that the UL142 luminal domain is involved in its cis-Golgi localization. We also confirm that UL142 down-modulates surface expression of full-length MICA alleles while having no effect on the truncated allele MICA*008. However, we demonstrate for the first time that UL142 retains full-length MICA alleles in the cis-Golgi apparatus. In addition, we propose that UL142 interacts with nascent MICA en route to the cell surface but not mature MICA at the cell surface. Our data also demonstrate that the UL142 luminal and transmembrane domains are involved in recognition and intracellular sequestration of full-length MICA alleles.

Immunosenescence and Cytomegalovirus: where do we stand after a decade?

Since Looney at al. published their seminal paper a decade ago it has become clear that many of the differences in T cell immunological parameters observed between young and old people are related to the age-associated increasing prevalence of infection with the persistent beta-herpesvirus HHV-5 (Cytomegalovirus). Ten years later, studies suggest that hallmark age-associated changes in peripheral blood T cell subset distribution may not occur at all in people who are not infected with this virus. Whether the observed changes are actually caused by CMV is an open question, but very similar, rapid changes observed in uninfected patients receiving CMV-infected kidney grafts are consistent with a causative role. This meeting intensively discussed these and other questions related to the impact of CMV on human immune status and its relevance for immune function in later life.

Intracellular Sequestration of the NKG2D Ligand ULBP3 by Human Cytomegalovirus

Human CMV (HCMV) encodes multiple genes that control NK cell activation and cytotoxicity. Some of these HCMV-encoded gene products modulate NK cell activity as ligands expressed at the cell surface that engage inhibitory NK cell receptors, whereas others prevent the infected cell from upregulating ligands that bind to activating NK cell receptors. A major activating NKR is the homodimeric NKG2D receptor, which has eight distinct natural ligands in humans. It was shown that HCMV is able to prevent the surface expression of five of these ligands (MIC A/B and ULBP1, 2, and 6). In this article, we show that the HCMV gene product UL142 can prevent cell surface expression of ULBP3 during infection. We further show that UL142 interacts with ULBP3 and mediates its intracellular retention in a compartment that colocalizes with markers of the cis-Golgi complex. In doing so, UL142 prevents ULBP3 trafficking to the surface and protects transfected cells from NK-mediated cytotoxicity. This is the first description of a viral gene able to mediate downregulation of ULBP3.