؉ memory CD8 T cells were found to persist in lung tissue. One can thus operationally distinguish an early CMV-positive IP (phase 1) and a late CMV-negative IP (phase 2). According to the definition, phase 2 histopathology would not be diagnosed as a CMV-IP and could instead be misinterpreted as a CMV-induced immunopathology. We document here that phase 1 as well as phase 2 pulmonary CD8 T cells are capable of exerting effector functions and are effectual in protecting against productive infection. We propose that antiviral "stand-by" memory-effector T cells persist in the lungs to prevent virus recurrence from latency.

Cytomegalovirus (CMV) infection in the period of temporary immunodeficiency after haematoablative treatment and bone marrow transplantation (BMT) is associated with a risk of graft failure and multiple-organ CMV disease. The efficacy of immune system reconstitution is decisive for the prevention of CMV pathogenesis after BMT. Previous data in murine model systems have documented a redundancy in the immune effector mechanisms controlling CMV. CD8 T cells proved to be relevant but not irreplaceable as antiviral effectors. Specifically, in a state of long-term in vivo depletion of the CD8 T-cell subset, CD4 T cells were educed to become deputy effectors controlling CMV by a mechanism involving antiviral cytokines. It is of medical importance to know whether one can trust in this ' flexible defence ' in all clinical settings. It is demonstrated here that reconstitution of CD8 T cells is crucial for the prevention of fatal multipleorgan CMV disease under the specific conditions of BMT.Patients undergoing bone marrow transplantation (BMT) associated with temporary immunodeficiency are at risk of fatal cytomegalovirus (CMV) disease with multiple organ involvement (for reviews see Winston et al., 1990 ; Einsele et al., 1998). Whether or not primary or recurrent CMV infections develop into disease appears to be related to the efficacy of haematopoietic and immune system reconstitution. Specifically, early reappearance of CD8 T cells is of positive prognostic value for recovery (Reusser et al., 1991). Accordingly, restoration of immunity to CMV by adoptive cytoimmunotherapy with antiviral CD8 T-cell lines is in clinical trials (Riddell et al., 1992 ;Walter et al., 1995). In BMT performed across minor or class I major histocompatibility disparities, graft vs. host disease (GvHD) is a further risk factor (Einsele et al., 1998). In a major Author for correspondence : Matthias Reddehase.Fax j49 6131 395604. e-mail Matthias.Reddehase!uni-mainz.de histocompatibility complex (MHC) class II-identical but MHC class I-disparate setting, selective depletion of class I-restricted CD8 T cells might be discussed as a regime for GvHD prophylaxis. However, if CD8 T cells were the only effectors Fig. 1. (A) Lethality of CMV infection after selective T-cell subset depletion. Syngeneic BMT was performed on day 0 with BALB/c mice as BM cell donors and recipients. Recipients were infected with 1i10 5 p.f.u. of purified murine CMV ca. 2 h after BMT. In vivo depletion of T-cell subsets was performed twice, namely on days 7 and 14, by intravenous administration of 1 mg of the respective antibody. Kaplan-Meyer plots show the survival rates for 20 recipients per experimental group as a function of time. (B) Efficacy and specificity of the T-cell subset depletions. Pulmonary infiltrate leukocytes were isolated on day 21 and analysed by three-colour cytofluorometry for the expression of TCR α/β (phycoerythrin fluorescence, FL-2), CD4 (RED613 fluorescence, FL-3) and CD8 (FITC fluorescence, FL-1). Gates were set on lymphocytes and on positive FL-2 in ...

The state of cytomegalovirus (CMV) after the resolution of acute infection is an unsolved problem in CMV research. While the term "latency" is in general use to indicate the maintenance of the viral genome, a formal exclusion of low-level persistent productive infection depends on the sensitivity of the assay for detecting infectious virus. We have improved the method for detecting infectivity by combining centrifugal infection of permissive indicator cells in culture, expansion to an infectious focus, and sensitive detection of immediateearly RNA in the infected cells by reverse transcriptase PCR. A limiting-dilution approach defined the sensitivity of this assay. Infectivity was thereby found to require as few as 2 to 9 virion DNA molecules of murine CMV, whereas the standard measure of infectivity, the PFU, is the equivalent of ca. 500 viral genomes. Since murine CMV forms multicapsid virions in most infected tissues, the genome-to-infectivity ratio is necessarily >1. This assay thus sets a new standard for investigating CMV latency. In mice in which acute infection was resolved, the viral DNA load in the lungs, a known organ site of CMV latency and recurrence, was found to be 1 genome per 40 lung cells, or a total of ca. 1 million genomes. Despite this high load of CMV DNA, infectious virus was not detected with the improved assay, but recurrence was inducible. These data provide evidence against a low-level persistent productive infection and also imply that intermittent spontaneous recurrence is not a frequent event in latently infected lungs.

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Interstitial pneumonia is a frequent and critical manifestation of human cytomegalovirus (CMV) disease in immunocompromised patients, in particular in recipients of bone marrow transplantation. Previous work in the murine CMV infection model has identified the lungs as a major organ site of CMV latency and recurrence. It was open to question whether the viral genome is transcriptionally silent or active during latency. Transcription could be latency associated and thus be part of the latency phenotype. Alternatively, transcriptional activity could reflect episodes of reactivation. We demonstrate here that transcription of the immediate-early (IE) transcription unitie1-ie3 selectively generatesie1-specific transcripts during latency. Notably, while the latent viral DNA was found to be evenly distributed in the lungs, transcription was focal and randomly distributed. This finding indicates that IE transcription is not a feature inherent to murine CMV latency but rather reflects foci of primordial reactivation. However, this reactivation did not initiate productive infection, sinceie3 gene mRNA specifying the essential transactivator IE3 of murine CMV early gene expression was not detectable. Accordingly, transcripts encoding gB were absent during latency. By contrast, during induced virus recurrence, IE-phase transcription switched from focal to generalized and ie3-specific transcripts were generated. These data imply that latency and recurrence are regulated not only at the IE promoter-enhancer and that there exists an additional checkpoint at the level of precursor RNA splicing. We propose that focal transcription reflects random episodes of nonproductive reactivation that get terminated before IE3 is expressed and ignites the productive cycle.