Andrew D. Hislop

Herpesvirus-Specific CD8 T Cell Immunity in Old Age: Cytomegalovirus Impairs the Response to a Coresident EBV Infection

Aging in humans is associated with increased infections and the reduced proliferative capacity of T cells, part of the more global phenomenon termed immune senescence. The etiology of immune senescence is unknown but the accumulation of virus-specific memory T cells may be a contributory factor. We have examined CD8 T cell responses to two persistent herpesvirus infections, CMV and EBV, and to a recurrent virus infection, influenza, in different age cohorts of healthy donors using HLA-peptide tetramers and intracellular cytokine detection. Of these, CMV appears to be the most immunogenic, with the CD8 T cell response representing over 10% of the CD8 pool in many elderly donors. Interestingly, the effect of age upon EBV-specific responses depends upon donor CMV sero-status. In CMV seropositive donors, the magnitude of the EBV-specific immune response is stable with age, but in CMV seronegative donors, the response to EBV increases significantly with age. By contrast, the influenza-specific CD8 T cell immune response decreases with age, independent of CMV status. The functional activity of the herpesvirus-specific immune response decreases in elderly donors, although the characteristic phenotypes of CMV- and EBV-specific memory populations are retained. This demonstrates that aging is associated with a marked accumulation of CMV-specific CD8 T cells together with a decrease in immediate effector function. Moreover, infection with CMV can reduce prevailing levels of immunity to EBV, another persistent virus. These results suggest that carriage of CMV may be detrimental to the immunocompetent host by suppressing heterologous virus-specific immunity during aging.

Epitope-specific Evolution of Human CD8+ T Cell Responses from Primary to Persistent Phases of Epstein-Barr Virus Infection

Primary virus infection often elicits a large CD8+ T cell response which subsequently contracts to a smaller memory T cell pool; the relationship between these two virus-specific populations is not well understood. Here we follow the human CD8+ T cell response to Epstein-Barr virus (EBV) from its primary phase in infectious mononucleosis (IM) through to the persistent carrier state. Using HLA-A2.1 or B8 tetramers specific for four lytic cycle and three latent cycle epitopes, we find marked differences in the epitope-specific composition of the T cell populations between the two phases of infection. The primary response is dominated by lytic epitope specificities which are severely culled (and in one case extinguished) with resolution of the acute infection; in contrast latent epitope specificities are less abundant, if present at all, in acute IM but often then increase their percentage representation in the CD8 pool. Even comparing epitopes of the same type, the relative size of responses seen in primary infection does not necessarily correlate with that seen in the longer term. We also follow the evolution of phenotypic change in these populations and show that, from a uniform CD45RA−RO+CCR7− phenotype in IM, lytic epitope responses show greater reversion to a CD45RA+RO− phenotype whereas latent epitope responses remain CD45RA−RO+ with a greater tendency to acquire CCR7. Interestingly these phenotypic distinctions reflect the source of the epitope as lytic or latent, and not the extent to which the response has been amplified in vivo.

EBV-Specific CD8+ T Cell Memory: Relationships Between Epitope Specificity, Cell Phenotype, and Immediate Effector Function

EBV infection in humans induces CD8+ T cell memory to viral epitopes derived from both lytic and latent cycle Ags. We have analyzed the relationship between the phenotype and function of the memory pool of T cells specific for these Ags. Lytic epitope-specific populations were heterogeneous in terms of CD45RO/RA and CD28 expression, whereas latent epitope-specific populations were uniformly CD45RO+ and CD28+, consistent with the higher antigenic challenge from lytic epitopes driving some memory cells toward a CD45RA+, CD28− phenotype. However, both types of memory population showed immediate epitope-specific cytotoxicity and type 1 cytokine production in ex vivo assays. Cytotoxic function was not associated with preactivated T cells, as EBV-specific populations were negative for activation markers such as CD69 or CD38, nor could cytotoxic function be ascribed to CD27− or CD56+ subsets, as such cells were not detected in EBV-specific memory. Furthermore, cytotoxicity was not limited to CD45RA+ and/or CD28− fractions, but also was observed in CD45RO+, CD28+ populations in lytic and latent epitope-specific memory. Cytokine (IFN-γ, TNF-α) responses, measured by intracytoplasmic staining after peptide stimulation, also were detectable in CD45RO+ and RA+ subsets as well as CD28+ and CD28− subsets. Of other markers that were heterogeneous in both lytic and latent epitope populations, CCR7 gave the best discrimination of functionality; thus, CCR7+ cells consistently failed to give an IFN-γ or TNF-α response, whereas many CCR7− cells were responsive. Our data are consistent with effector functions having a broad distribution among phenotypically distinct subsets of “effector memory” cells that have lost the CCR7 marker.

Memory T Cells Constitute a Subset of the Human CD8+CD45RA+ Pool with Distinct Phenotypic and Migratory Characteristics

Using HLA class I-viral epitope tetramers to monitor herpes virus-specific CD8+ T cell responses in humans, we have shown that a significant fraction of responding cells revert from a CD45RO+ to a CD45RA+ state after priming. All tetramer-binding CD45RA+ cells, regardless of epitope specificity, expressed a phenotype LFA-1highCCR7low that was stable for at least 10 years in infectious mononucleosis patients and indefinitely in asymptomatic carriers. CD8+CD45RA+LFA-1high cells were not present in cord blood but in adults account for up to 50% of CD8+CD45RA+ cells. These CD45RA+LFA-1high cells have significantly shorter telomeres than CD45RA+LFA-1low cells, suggesting that the latter represent a naive population, while the former are memory cells. CD45RA+ memory cells are a stable population of noncycling cells, but on stimulation they are potent producers of IFN-γ, while naive CD8+ cells produce only IL-2. The chemokine receptor profile and migratory potential of CD45RA+ memory cells is very similar to CD45RO+ cells but different to naive CD8 cells. In accord with this, CD45RA+ memory cells were significantly underrepresented in lymph nodes, but account for virtually all CD8+CD45RA+ T cells in peripheral tissues of the same individuals.

A CD8+ T cell immune evasion protein specific to Epstein-Barr virus and its close relatives in Old World primates

γ1-Herpesviruses such as Epstein-Barr virus (EBV) have a unique ability to amplify virus loads in vivo through latent growth-transforming infection. Whether they, like α- and β-herpesviruses, have been driven to actively evade immune detection of replicative (lytic) infection remains a moot point. We were prompted to readdress this question by recent work (Pudney, V.A., A.M. Leese, A.B. Rickinson, and A.D. Hislop. 2005. J. Exp. Med. 201:349–360; Ressing, M.E., S.E. Keating, D. van Leeuwen, D. Koppers-Lalic, I.Y. Pappworth, E.J.H.J. Wiertz, and M. Rowe. 2005. J. Immunol. 174:6829–6838) showing that, as EBV-infected cells move through the lytic cycle, their susceptibility to EBV-specific CD8+ T cell recognition falls dramatically, concomitant with a reductions in transporter associated with antigen processing (TAP) function and surface human histocompatibility leukocyte antigen (HLA) class I expression. Screening of genes that are unique to EBV and closely related γ1-herpesviruses of Old World primates identified an early EBV lytic cycle gene, BNLF2a, which efficiently blocks antigen-specific CD8+ T cell recognition through HLA-A–, HLA-B–, and HLA-C–restricting alleles when expressed in target cells in vitro. The small (60–amino acid) BNLF2a protein mediated its effects through interacting with the TAP complex and inhibiting both its peptide- and ATP-binding functions. Furthermore, this targeting of the major histocompatibility complex class I pathway appears to be conserved among the BNLF2a homologues of Old World primate γ1-herpesviruses. Thus, even the acquisition of latent cycle genes endowing unique growth-transforming ability has not liberated these agents from evolutionary pressure to evade CD8+ T cell control over virus replicative foci.

Increased Expression of the NK Cell Receptor KLRG1 by Virus-Specific CD8 T Cells during Persistent Antigen Stimulation

ABSTRACT The killer cell lectin-like receptor G1 (KLRG1) is a natural killer cell receptor expressed by T cells that exhibit impaired proliferative capacity. Here, we determined the KLRG1 expression by virus-specific T cells. We found that repetitive and persistent antigen stimulation leads to an increase in KLRG1 expression of virus-specific CD8+ T cells in mice and that virus-specific CD8+ T cells are mostly KLRG1+ in chronic human viral infections (human immunodeficiency virus, cytomegalovirus, and Epstein-Barr virus) but not in resolved infection (influenza virus). Thus, by using KLRG1 as a T-cell marker, our results suggest that the differentiation status and function of virus-specific CD8+ T cells are directly influenced by persistent antigen stimulation.

CD8+ immunodominance among Epstein-Barr virus lytic cycle antigens directly reflects the efficiency of antigen presentation in lytically infected cells

Antigen immunodominance is an unexplained feature of CD8+ T cell responses to herpesviruses, which are agents whose lytic replication involves the sequential expression of immediate early (IE), early (E), and late (L) proteins. Here, we analyze the primary CD8 response to Epstein-Barr virus (EBV) infection for reactivity to 2 IE proteins, 11 representative E proteins, and 10 representative L proteins, across a range of HLA backgrounds. Responses were consistently skewed toward epitopes in IE and a subset of E proteins, with only occasional responses to novel epitopes in L proteins. CD8+ T cell clones to representative IE, E, and L epitopes were assayed against EBV-transformed lymphoblastoid cell lines (LCLs) containing lytically infected cells. This showed direct recognition of lytically infected cells by all three sets of effectors but at markedly different levels, in the order IE > E ≫ L, indicating that the efficiency of epitope presentation falls dramatically with progress of the lytic cycle. Thus, EBV lytic cycle antigens display a hierarchy of immunodominance that directly reflects the efficiency of their presentation in lytically infected cells; the CD8+ T cell response thereby focuses on targets whose recognition leads to maximal biologic effect.

The Immunology of Epstein-Barr Virus–Induced Disease

Epstein-Barr virus (EBV) is usually acquired silently early in life and carried thereafter as an asymptomatic infection of the B lymphoid system. However, many circumstances disturb the delicate EBV-host balance and cause the virus to display its pathogenic potential. Thus, primary infection in adolescence can manifest as infectious mononucleosis (IM), as a fatal illness that magnifies the immunopathology of IM in boys with the X-linked lymphoproliferative disease trait, and as a chronic active disease leading to life-threatening hemophagocytosis in rare cases of T or natural killer (NK) cell infection. Patients with primary immunodeficiencies affecting the NK and/or T cell systems, as well as immunosuppressed transplant recipients, handle EBV infections poorly, and many are at increased risk of virus-driven B-lymphoproliferative disease. By contrast, a range of other EBV-positive malignancies of lymphoid or epithelial origin arise in individuals with seemingly intact immune systems through mechanisms that remain to be understood.

Tonsillar homing of Epstein-Barr virus–specific CD8+ T cells and the virus-host balance

Patients with infectious mononucleosis (IM) undergoing primary EBV infection show large expansions of EBV-specific CD8+ T cells in the blood. While latent infection of the B cell pool is quickly controlled, virus shedding from lytically infected cells in the oropharynx remains high for several months. We therefore studied how responses localize to the tonsil, a major target site for EBV, during primary infection and persistence. In acute IM, EBV-specific effectors were poorly represented among CD8+ T cells in tonsil compared with blood, coincident with absence of the CCR7 lymphoid homing marker on these highly activated cells. In patients who had recently recovered from IM, latent epitope reactivities were quicker than lytic reactivities both to acquire CCR7 and to accumulate in the tonsil, with some of these cells now expressing the CD103 integrin, which mediates retention at mucosal sites. By contrast, in long-term virus carriers in whom both lytic and latent infections had been controlled, there was 2- to 5-fold enrichment of lytic epitope reactivities and 10- to 20-fold enrichment of latent epitope reactivities in tonsil compared with blood; up to 20% of tonsillar CD8+ T cells were EBV specific, and many now expressed CD103. We suggest that efficient control of EBV infection requires appropriate CD8+ T cell homing to oropharyngeal sites.

Molecular Pathogenesis of EBV Susceptibility in XLP as Revealed by Analysis of Female Carriers with Heterozygous Expression of SAP

Analysis of females carriers of the X-linked lymphoproliferative (XLP) trait reveals the mechanism underlying exquisite sensitivity of XLP patients to often-fatal infection with the normally innocuous Epstein-Barr virus.