Characterization of the human EBV-specific CD4+ T cell response using MHC II tetramers reveals the latent EBV antigen response is more frequent than the lytic response with a delayed EBNA1 response that coincides with diminished cross-presentation.

The complement regulatory protein decay accelerating factor (DAF; CD55), inhibits the alternative complement pathway by accelerating decay of the convertase enzymes formed by C3b and factor B. We show, using surface plasmon resonance, that in the absence of Mg 2؉ , DAF binds C3b, factor B, and the Bb subunit with low affinity (K D , 14 ؎ 0.1, 44 ؎ 10, and 20 ؎ 7 M, respectively). In the presence of Mg 2؉ , DAF bound Bb or the von Willebrand factor type A subunit of Bb with higher affinities (K D , 1.3 ؎ 0.5 and 2.2 ؎ 0.1 M, respectively). Interaction with the proenzyme C3bB was investigated by flowing factor B across a C3b-coated surface in the absence of factor D. The dissociation rate was dependent on the time of incubation, suggesting that a time-dependent conformational transition stabilized the C3b-factor B interaction. Activation by factor D (forming C3bBb) increased the complex half-life; however, the enzyme became susceptible to rapid decay by DAF, unlike the proenzyme, which was unaffected. A convertase assembled with cobra venom factor and Bb was decayed by DAF, albeit far less efficiently than C3bBb. DAF did not bind cobra venom factor, implying that Bb decay is accelerated, at least in part, through DAF binding of this subunit. It is likely that DAF binds the complex with higher affinity/avidity, promoting a conformational change in either or both subunits accelerating decay. Such analysis of component and regulator interactions will inform our understanding of inhibitory mechanisms and the ways in which regulatory proteins cooperate to control the complement cascade.

CD97, the archetypal member of the EGF-TM7 protein family, is constitutively expressed on granulocytes and monocytes and rapidly up-regulated on T and B cells following activation. The key isoform of CD97 expressed on leukocytes binds the complement regulatory protein CD55 (also termed decay-accelerating factor). CD97 has been shown recently to mediate costimulation of T cells via CD55. Here, we demonstrate that blocking the interaction between CD55 on monocytes and CD97 on T cells leads to inhibition of proliferation and interferon-␥ secretion. This implies that bidirectional interactions between CD97 and CD55 are involved in T cell regulation. Structural studies presented here reveal the molecular basis for this activity. We have solved the structure of EMR2, a very close homolog of CD97, using x-ray crystallography. NMR-based chemical shift mapping of the EMR2-CD55 interaction has allowed us to generate a model for the CD97-CD55 complex. The structure of the complex reveals that the T cell and complement regulatory activities of CD55 occur on opposite faces of the molecule. This suggests that CD55 might simultaneously regulate both the innate and adaptive immune responses, and we have shown that CD55 can still regulate complement when bound to CD97.

In patients with XLP, a primary immunodeficiency caused by mutations in SH2D1A, EBV infection can lead to somatic reversion of the disease-causing mutation selectively in effector memory CD8 T cells; reverted CD8 cells are better able to respond to and kill EBV-infected cells.

Epstein-Barr virus (EBV) is typically acquired asymptomatically in childhood. In contrast, infection later in life often leads to infectious mononucleosis (IM), a febrile illness characterized by anti-EBV IgM antibody positivity, high loads of circulating latently infected B cells, and a marked lymphocytosis caused by hyperexpansion of EBV-specific CD8+ T cells plus a milder expansion of CD56dim NKG2A+ KIR− natural killer (NK) cells. How the two situations compare is unclear due to the paucity of studies on clinically silent infection. Here we describe five prospectively studied patients with asymptomatic infections identified in a seroepidemiologic survey of university entrants. In each case, the key blood sample had high cell-associated viral loads without a marked CD8 lymphocytosis or NK cell disturbance like those seen in patients during the acute phase of IM. Two of the cases with the highest viral loads showed a coincident expansion of activated EBV-specific CD8+ T cells, but overall CD8+ T cell numbers were either unaffected or only mildly increased. Two cases with slightly lower loads, in whom serology suggests the infection may have been caught earlier in the course of infection, also showed no T or NK cell expansion at the time. Interestingly, in another case with a higher viral load, in which T and NK cell responses were undetectable in the primary blood sample in which infection was detected, EBV-specific T cell responses did not appear until several months later, by which time the viral loads in the blood had already fallen. Thus, some patients with asymptomatic primary infections have very high circulating viral loads similar to those in patients during the acute phase of IM and a cell-mediated immune response that is qualitatively similar to that in IM patients but of a lower magnitude. However, other patients may have quite different immune responses that ultimately could reveal novel mechanisms of host control.IMPORTANCE Epstein-Barr virus (EBV) is transmitted orally, replicates in the throat, and then invades the B lymphocyte pool through a growth-transforming latent infection. While primary infection in childhood is usually asymptomatic, delayed infection is associated with infectious mononucleosis (IM), a febrile illness in which patients have high circulating viral loads and an exaggerated virus-induced immune response involving both CD8+ T cells and natural killer (NK) cells. Here we show that in five cases of asymptomatic infection, viral loads in the blood were as high as those in patients during the acute phase of IM, whereas the cell-mediated responses, even when they resembled those in patients during the acute phase of IM in timing and quality, were never as exaggerated. We infer that IM symptoms arise as a consequence not of the virus infection per se but of the hyperactivated immune response. Interestingly, there were idiosyncratic differences among asymptomatic cases in the relationship between the viral load and the response kinetics, emphasizing how much there is still to ...

Using double electron electron resonance (DEER) spectroscopy, a distance of 6.15 nm is measured between the nitroxide labels of von Willebrand Factor A domains. The picture shows the DEER spectrum and the trimeric structure of the protein that is deduced from the DEER results. For more information see the Communication by G. Jeschke and co-workers on the following pages.

CD8+ T cell responses to Epstein-Barr virus (EBV) lytic cycle expressed antigens display a hierarchy of immunodominance, in which responses to epitopes of immediate-early (IE) and some early (E) antigens are more frequently observed than responses to epitopes of late (L) expressed antigens. It has been proposed that this hierarchy, which correlates with the phase-specific efficiency of antigen presentation, may be due to the influence of viral immune-evasion genes. At least three EBV-encoded genes, BNLF2a, BGLF5 and BILF1, have the potential to inhibit processing and presentation of CD8+ T cell epitopes. Here we examined the relative contribution of these genes to modulation of CD8+ T cell recognition of EBV lytic antigens expressed at different phases of the replication cycle in EBV-transformed B-cells (LCLs) which spontaneously reactivate lytic cycle. Selective shRNA-mediated knockdown of BNLF2a expression led to more efficient recognition of immediate-early (IE)- and early (E)-derived epitopes by CD8+ T cells, while knock down of BILF1 increased recognition of epitopes from E and late (L)-expressed antigens. Contrary to what might have been predicted from previous ectopic expression studies in EBV-negative model cell lines, the shRNA-mediated inhibition of BGLF5 expression in LCLs showed only modest, if any, increase in recognition of epitopes expressed in any phase of lytic cycle. These data indicate that whilst BNLF2a interferes with antigen presentation with diminishing efficiency as lytic cycle progresses (IE>E>>L), interference by BILF1 increases with progression through lytic cycle (IE

Epstein-Barr virus (EBV) elicits primary CD8+ T cell responses that, by T cell cloning from infectious mononucleosis (IM) patients, appear skewed towards immediate early (IE) and some early (E) lytic cycle proteins, with late (L) proteins rarely targeted. However, L antigen-specific responses have been regularly detected in polyclonal T cell cultures from long-term virus carriers. To resolve this apparent difference between responses to primary and persistent infection, 13 long-term carriers were screened in ex vivo IFN-γ ELISPOT assays using peptides spanning the 2 IE, 6 representative E and 7 representative L proteins. This revealed memory CD8 responses to 44 new lytic cycle epitopes that straddle all three protein classes but, in terms of both frequency and size, maintain the IE > E > L hierarchy of immunodominance. Having identified the HLA restriction of 10 (including 7L) new epitopes using memory CD8+ T cell clones, we looked in HLA-matched IM patients and found such reactivities but typically at low levels, explaining why they had gone undetected in the original IM clonal screens. Wherever tested, all CD8+ T cell clones against these novel lytic cycle epitopes recognised lytically-infected cells naturally expressing their target antigen. Surprisingly, however, clones against the most frequently recognised L antigen, the BNRF1 tegument protein, also recognised latently-infected, growth-transformed cells. We infer that BNRF1 is also a latent antigen that could be targeted in T cell therapy of EBV-driven B-lymphoproliferative disease.