IL-7R alpha versus CCR7 and CD45 as markers of virus-specific CD8+ T cell differentiation: contrasting pictures in blood and tonsillar lymphoid tissue.

In humans, circulating CD8(+) memory T cells to a nonpersistent virus (influenza) lie within CCR7(+)CD45RA(-) central memory, whereas memory to Epstein-Barr virus (EBV) latent, EBV lytic, and cytomegalovirus (CMV) antigens are progressively larger in size and are more biased toward CCR7(-)CD45RA(-) effector memory and CCR7(-)CD45RA(+) terminally differentiated compartments. We found that these populations are also distinguished by progressively lower expression of the interleukin-7 receptor (IL-7R alpha) and by lower IL-7 responsiveness; indeed, percentage IL-7R alpha -positive values showed a tight inverse correlation with population size. However, these relationships among size, differentiation phenotype, and IL-7R alpha status in blood did not hold in tonsillar tissue. In tonsil tissue, although EBV reactivities outnumbered their CMV and influenza counterparts, the distinct CCR7/CD45 isoform signatures of the different virus-specific populations were retained. Moreover, all detectable reactivities showed high levels of IL-7R alpha expression. As a discriminator between different virus-specific populations, IL-7R alpha therefore appears to be more susceptible to tissue location than the classical CCR7/CD45 markers.

Cytotoxic T-lymphocyte responses to a polymorphic Epstein-Barr virus epitope identify healthy carriers with coresident viral strains.

Cytotoxic T-lymphocyte (CTL) responses to Epstein-Barr virus (EBV) tend to focus on a few immunodominant viral epitopes; where these epitope sequences are polymorphic between EBV strains, host CTL specificities should reflect the identity of the resident strain. In studying responses in HLA-B27-positive virus carriers, we identified 2 of 15 individuals who had strong CTL memory to the pan-B27 epitope RRIYDLIEL (RRIY) from nuclear antigen EBNA3C but whose endogenous EBV strain, isolated in vitro, encoded a variant sequence RKIYDLIEL (RKIY) which did not form stable complexes with B27 molecules and which was poorly recognized by RRIY-specific CTLs. To check if such individuals were also carrying an epitope-positive strain (either related to or distinct from the in vitro isolate), we screened DNA from freshly isolated peripheral blood mononuclear cells for amplifiable virus sequences across the EBNA3C epitope, across a different region of EBNA3C with type 1-type 2 sequence divergence, and across a polymorphic region of EBNA1. This showed that one of the unexplained RRIY responders carried two distinct type 1 strains, one with an RKIY and one with an RRIY epitope sequence. The other responder carried an RKIY-positive type 1 strain and a type 2 virus whose epitope sequence of RRIFDLIEL was antigenically cross-reactive with RRIY. Of 15 EBV-seropositive donors analyzed by such assays, 12 appeared to be carrying a single virus strain, one was coinfected with distinct type 1 strains, and two were carrying both type 1 and type 2 viruses. This implies that a small but significant percentage of healthy virus carriers harbor multiple, perhaps sequentially acquired, EBV strains.

HLA-B27 subtype polymorphism and CTL epitope choice: studies with EBV peptides link immunogenicity with stability of the B27:peptide complex.

HLA-B27-restricted CTL responses to EBV are principally directed against two of the EBV nuclear Ags, EBNAs 3B and 3C. We have previously described a target epitope derived from EBNA 3C (residues 258-266, sequence RRIYDLIEL) that is immunodominant in the context of at least three different B27 subtypes, including B*2705 and B*2702. In this study, we show that this peptide binds well to B*2705 and B*2702 in a cell surface binding assay, and that the two B27:peptide complexes are relatively stable, with t1/2 of 20 and 37 h, respectively. We now identify another B27-restricted epitope derived from EBNA 3B (residues 243-253, sequence RRARSLSAERY), which again accords well with the B*2705/B*2702 consensus motifs, having an arginine residue at position 2 and a tyrosine residue at the carboxyl terminus. In this case, five of five B*2702-positive donors respond to the epitope, whereas there was no response in any B*2705-positive donor studied. This peptide binds at least as well to B*2705 as to its restriction element B*2702; however, the two class I:peptide complexes show marked differences in stability, with t1/2 of 9 and 42 h, respectively. Thus, the stability of B27:peptide complexes can vary markedly between different B27 subtypes in ways that may not be apparent from cell surface binding assays and cannot be predicted from currently known peptide consensus motifs, yet which may critically influence CTL epitope choice.

Immediate early and early lytic cycle proteins are frequent targets of the Epstein-Barr virus-induced cytotoxic T cell response.

Epstein-Barr virus (EBV), a human gamma-herpesvirus, can establish both nonproductive (latent) and productive (lytic) infections. Although the CD8+ cytotoxic T lymphocyte (CTL) response to latently infected cells is well characterized, very little is known about T cell controls over lytic infection; this imbalance in our understanding belies the importance of virus-replicative lesions in several aspects of EBV disease pathogenesis. The present work shows that the primary CD8+ CTL response to EBV in infectious mononucleosis patients contains multiple lytic antigen-specific reactivities at levels at least as high as those seen against latent antigens; similar reactivities are also detectable in CTL memory. Clonal analysis revealed individual responses to the two immediate early proteins BZLF1 and BRLF1, and to three (BMLF1, BMRF1, and BALF2) of the six early proteins tested. In several cases, the peptide epitope and HLA-restricting determinant recognized by these CTLs has been defined, one unusual feature being the number of responses restricted through HLA-C alleles. The work strongly suggests that EBV-replicative lesions are subject to direct CTL control in vivo and that immediate early and early proteins are frequently the immunodominant targets. This contrasts with findings in alpha- and beta-herpesvirus systems (herpes simplex, cytomegalovirus) where viral interference with the antigen-processing pathway during lytic infection renders immediate early and early proteins much less immunogenic. The unique capacity of gamma-herpesvirus to amplify the viral load in vivo through a latent growth-transforming infection may have rendered these agents less dependent upon viral replication as a means of successfully colonizing their hosts.

Epitope focusing in the primary cytotoxic T cell response to Epstein-Barr virus and its relationship to T cell memory.

The relationship between primary and memory cytotoxic T lymphocyte (CTL) responses, and the factors influencing entry into memory, are poorly understood. Here we address this in the context of Epstein-Barr virus (EBV), a persistent human herpesvirus in which memory CTL responses in long-term virus carriers are highly focused on epitopes preferentially drawn from just three of the eight available virus latent proteins, EBNAs 3A, 3B, and 3C. To determine whether this unusual level of focusing is a consequence of long-term virus challenge, we carried out a detailed analysis of EBV antigen/epitope specificities in the primary virus-induced CTL response in 10 infectious mononucleosis (IM) patients of different HLA types. Primary effectors, studied in ex vivo assays and by limiting dilution cloning in vitro, were again highly skewed toward a small number of viral epitopes, almost all derived from the EBNA3 proteins, with CTL to the immunodominant epitope accounting for at least 1% of the circulating CD8+ IM T cell pool. This is the first unequivocal demonstration of an EBV-specific CD8+ CTL response in IM. Prospective studies on individual patients showed that, whereas all of the EBV reactivities found in CTL memory had been detectable earlier during primary infection, the memory population was not simply a scaled down version of the primary response. In particular (a) differences in the relative frequencies of CTL to immunodominant versus subdominant epitopes appeared to be much less marked in memory than in primary populations, and (b) we found at least one clear example in which a significant virus-specific reactivity within the primary response was never detectable in memory.

Estimation of oestrogen and progesterone receptors in chronic rhinitis.

The turbinates of 38 patients with chronic rhinitis were examined biochemically for oestrogen and progesterone receptors. Low levels of oestrogen-receptor-like activity (1-20 fmol/mg protein) were found in 50% of patients of both sexes. Progesterone receptor activity was also weak (1-16 fmol/mg protein) but was present only in 5 female patients. Immunocytochemical assay failed to demonstrate focal areas of oestrogen receptor activity. One juvenile nasopharyngeal angiofibroma was negative for both oestrogen and androgen receptors. Other possible mechanisms of hormonal action are considered.

A survey of the non-attendance rate at the ENT clinic of a district general hospital.

A survey of the number of patients who failed to keep outpatient appointments at the ENT clinic of a rurally situated district general hospital (Bangour General Hospital, West Lothian) was carried out. The results were compared with non-attendance rates of the general medical clinic of the same hospital and of the ENT clinic of a nearby urban hospital (the Royal Infirmary in Edinburgh). A particularly high non-attendance rate was demonstrated for the ENT clinic of the district general hospital. The reasons for this are discussed in the light of currently available literature on the 'no show' patient and possible means of improving the situation are suggested.