. 74:7496-7507, 2000). It was proposed that these cells represent antiviral "standby" memory cells whose functional role might be to help prevent reactivation of latent virus. The pool of pulmonary CD8 T cells was composed of two subsets defined by the T-cell activation marker L-selectin (CD62L): a CD62Lhi subset of quiescent memory cells, and a CD62L lo subset of recently resensitized memory-effector cells. In this study, we have continued this line of investigation by quantitating CD8 T cells specific for the three currently published antigenic peptides of mCMV: peptide YPHFMPTNL processed from the immediate-early protein IE1 (pp89), and peptides YGPSLYRRF and AYAGLFTPL, derived from the early proteins m04 (gp34) and M84 (p65), respectively. IE1-specific CD8 T cells dominated in acute-phase pulmonary infiltrates and were selectively enriched in latently infected lungs. Notably, most IE1-specific CD8 T cells were found to belong to the CD62L lo subset representing memory-effector cells. This finding is in accordance with the interpretation that IE1-specific CD8 T cells are frequently resensitized during latent infection of the lungs and may thus be involved in the maintenance of mCMV latency.In human cytomegalovirus (hCMV) infection after bone marrow transplantation (BMT), recovery from CMV disease correlates with efficient reconstitution of CD8 T cells (50). Preemptive cytoimmunotherapy by adoptive transfer of hCMVspecific CD8 T-cell clones was found to be beneficial in that it reduced the incidence of CMV disease in BMT recipients (51, 56). Proof of principle for the protective effect of antiviral CD8 T cells was provided by the model of murine CMV (mCMV) infection of BALB/c mice subjected to hematoablative treatment. Early experiments performed in the absence of BMT documented an antiviral and protective function of adoptively transferred mCMV-specific CD8 T cells in the lungs as well as in other target organs of the disease (44, 46, 48; for a review, see reference 23). More recently, the course of mCMV infection was analyzed in the specific context of hematolymphopoietic reconstitution after either syngeneic BMT (18,38,39) or BMT performed across a single major histocompatibility complex (MHC) class I antigen disparity (1). Prevention of a disseminated and fulminant interstitial CMV pneumonia by the antiviral function of endogenously reconstituted CD8 T cells was inferred from the following observations: (i) CD8 T cells rather than CD4 T cells were recruited to infected lungs much more efficiently than to uninfected lungs (18); (ii) lung-infiltrating, blastoid CD62L lo CD8 T cells were not randomly distributed in lung tissue but were found to colocalize with infected lung cells in inflammatory foci, thereby secluding the infected cells from health tissue (18, 38); (iii) when isolated from the infiltrates, these activated CD8 T cells exerted ex vivo cytolytic activity against infected target cells (18) and secreted gamma interferon (IFN-␥) upon polyclonal triggering via CD3ε (38); (iv) the kinetics of infilt...

؉ 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.

Cytomegaloviruses (CMVs) code for several proteins that inhibit the presentation of antigenic peptides to CD8 T cells. Although the molecular mechanisms of CMV interference with the major histocompatibility complex class I pathway are long understood, surprisingly little evidence exists to support a role in vivo. Here we document the first example of the presentation of an antigenic peptide being blocked by a CMV immune evasion protein in organs relevant to CMV disease. Although this D b -restricted peptide, which is derived from the antiapoptotic protein M45 of murine CMV (mCMV), is classified as an immunodominant peptide based on response magnitude and long-term memory, adoptive transfer of M45 epitope-specific CD8 T cells did not protect against infection with wild-type mCMV. Notably, the same cells protected C57BL/6 mice infected with an mCMV mutant in which immune evasion protein m152/gp40 is deleted. These data indicate that direct presentation or cross-presentation of an antigenic peptide by professional antigen-presenting cells can efficiently prime CD8 T cells that fail in protection against CMV organ disease because m152/gp40 prevents presentation of this peptide in pathogenetically relevant tissue cells.

Reconstitution of antiviral CD8 T cells is essential for controlling cytomegalovirus (CMV) infectionThe L d -restricted immediate-early 1 (IE1) peptide 168-YPHFMPTNL-176 of murine cytomegalovirus (mCMV) was the first antigenic peptide to be identified for a herpesvirus (50). The IE1 protein derived from open reading frame (ORF) m123, an intranuclear phosphoprotein which exists in molecular species of 89 and 76 kDa (32), is expressed in the IE phase of viral gene expression and performs regulatory and transactivating functions (12,31,39). It is encoded in transcription unit ie1/3 of which mRNAs specifying proteins IE1 (encoded by exons 2, 3, and 4) and IE3 (encoded by exons 2, 3, and 5) are generated by differential splicing (31, 39). The IE1 protein is processed to yield peptide 168-YPHFMPTNL-176 and an Nterminally elongated precursor 166-DMYPHFMPTNL-176 by the constitutive proteasome and, more efficiently, by the immunoproteasome, with only the precursor being translocated into the lumen of the endoplasmic reticulum for major histocompatibility complex class I (MHC-I) loading (36). N-terminal trimming finally leads to the IE1 peptide presented by the MHC-I molecule L d (reviewed in reference 45). Based on the frequency of IE1 epitope-specific CD8 T cells primed during acute infection and on the establishment of long-term IE1-specific memory, the IE1 peptide was classified as one of just two immunodominant MHC-I-restricted antigenic peptides in the H-2 d haplotype (25). Owing to MHC polymorphism, it is evident that immunodominance of peptides and the proteins from which they are derived cannot be extrapolated to other haplotypes in the same animal species. This is all the more true for extrapolation from mCMV to antigenic peptides of human cytomegalovirus (hCMV) presented by HLA molecules. Thus, although an early report by Borysiewicz et al. (4) had indicated the CD8 T-cell immunogenicity of the hCMV IE1 ortholog, the predictive value of the BALB/c mouse model in terms of the role of IE proteins in immunity to CMVs has been debated for a long time, until more recently the IE1 immunogenicity in humans was revisited with unbiased new methodology (10,14,33,34). Most intriguingly, in a comprehensive pangenomic search for antigenic ORFs of hCMV by using overlapping peptides and by covering all major HLA molecules present in the human population, Louis Picker and colleagues identified ORF UL123 encoding the IE1 protein as one of the top three antigen-encoding ORFs of hCMV that are most frequently detected by HLA class I-restricted human CD8 T cells (L. Picker, "T cell recognition of hCMV in natural human infection: pan-genome analysis of immunogenic open reading frames," Instituto Juan March de Estudios e Investigaciones workshop "Immunodominance: the key to understanding and manipulating CD8 ϩ T cell responses

CD8 T cells are the principal antiviral effectors controlling cytomegalovirus (CMV) infection.For human CMV, the virion tegument protein ppUL83 (pp65) has been identified as a source of immunodominant peptides and is regarded as a candidate for cytoimmunotherapy and vaccination. Two sequence homologs of ppUL83 are known for murine CMV, namely the virion protein ppM83 (pp105) expressed late in the viral replication cycle and the nonstructural protein pM84 (p65) expressed in the early phase. Here we show that ppM83, unlike ppUL83, is not delivered into the antigen presentation pathway after virus penetration before or in absence of viral gene expression, while other virion proteins of murine CMV are processed along this route. In cytokine secretion-based assays, ppM83 and pM84 appeared to barely contribute to the acute immune response and to immunological memory. Specifically, the frequencies of M83 and M84 peptide-specific CD8 T cells were low and undetectable, respectively. Nonetheless, in a murine model of cytoimmunotherapy of lethal CMV disease, M83 and M84 peptide-specific cytolytic T-cell lines proved to be highly efficient in resolving productive infection in multiple organs of cell transfer recipients. These findings demonstrate that proteins which fail to prime a quantitatively dominant immune response can nevertheless represent relevant antigens in the effector phase. We conclude that quantitative and qualitative immunodominance are not necessarily correlated. As a consequence of these findings, there is no longer a rationale for considering T-cell abundance as the key criterion for choosing specificities to be included in immunotherapy and immunoprophylaxis of CMV disease and of viral infections in general.Resolution of human cytomegalovirus (hCMV) infection after bone marrow transplantation (BMT) correlates with the reconstitution of CD8 T cells (54). Clinical trials of cytoimmunotherapy in BMT patients gave promising results in that preemptively transferred hCMV-specific CD8 T-cell clones colonized and persisted for a long time in the recipients and, to the best of our knowledge, did not exert adverse immunopathological effects (56). Since hCMV infection and disease were rarely observed in the recipients, it was justified to propose an antiviral, protective effect of the transferred cells (56,64). Although the hCMV genome has coding capacity for as many as ca. 200 proteins, only few of those are thought to be relevantly involved in the antiviral control by CD8 T cells (for a recent review, see reference 44). The list so far includes the virion tegument proteins ppUL32 (pp150 or basic phosphoprotein) and ppUL83 (pp65 or lower matrix protein), the virion envelope glycoprotein gpUL55 (gB), and the nonvirion regulatory immediate-early (IE) protein ppUL123 (IE1 or pp68-72). There are no evident common features that could explain the immunodominance of this limited set of proteins, but one may speculate that viral immune evasion mechanisms interfering with the major histocompatibility complex (MHC) class I ...

Preclinical research in murine models as well as subsequent clinical trials have concordantly revealed a high protective potential of antiviral CD8 T cells, of donorderived ex vivo memory CD8 T cells in particular, in the immunotherapy of cytomegalovirus (CMV) infection in immunocompromised recipients. Although it is generally held view that the observed beneficial effect of the transferred cells is viral epitope-specific, involving the recognition of MHC class-I presented peptides by cognate T cell receptors, this assumption awaits formal proof, at least with regard to the in vivo function of the CD8 T cells. This question is particularly evident for CMV, since the function of viral immune evasion proteins interferes with the MHC class-I pathway of peptide presentation. Alternatively, therefore, one has to consider the possibility that the requirement for epitope recognition may be bypassed by other ligand-receptor interactions between CD8 T cells and infected cells, which may trigger the signaling for effector functions. Clearly, such a mechanism might explain why CD8 T cells are so efficient in controlling CMV infection despite the expression of viral immune evasion proteins. Here we provide direct evidence for epitope-specificity of antiviral protection by employing a recombinant murine CMV (mCMV), namely the mutant virus mCMV-IE1-L176A, in which an immunodominant viral epitope of the regulatory immediate-early protein IE1 is functionally deleted by a point mutation replacing leucine with alanine at the C-terminal MHC anchor position of the antigenic peptide.