Mast cells expedite control of pulmonary murine cytomegalovirus infection by enhancing the recruitment of protective CD8 T cells to the lungs.

The lungs are a noted predilection site of acute, latent, and reactivated cytomegalovirus (CMV) infections. Interstitial pneumonia is the most dreaded manifestation of CMV disease in the immunocompromised host, whereas in the immunocompetent host lung-infiltrating CD8 T cells confine the infection in nodular inflammatory foci and prevent viral pathology. By using murine CMV infection as a model, we provide evidence for a critical role of mast cells (MC) in the recruitment of protective CD8 T cells to the lungs. Systemic infection triggered degranulation selectively in infected MC. The viral activation of MC was associated with a wave of CC chemokine ligand 5 (CCL5) in the serum of C57BL/6 mice that was MC-derived as verified by infection of MC-deficient Kit(W-sh/W-sh) "sash" mutants. In these mutants, CD8 T cells were recruited less efficiently to the lungs, correlating with enhanced viral replication and delayed virus clearance. A causative role for MC was verified by MC reconstitution of "sash" mice restoring both, efficient CD8 T-cell recruitment and infection control. These results reveal a novel crosstalk axis between innate and adaptive immune defense against CMV, and identify MC as a hitherto unconsidered player in the immune surveillance at a relevant site of CMV disease.

Identification of an atypical CD8 T cell epitope encoded by murine cytomegalovirus ORF-M54 gaining dominance after deletion of the immunodominant antiviral CD8 T cell specificities.

Control of murine cytomegalovirus (mCMV) infection is mediated primarily by CD8 T cells, with four specificities dominating in BALB/c mice. Functional deletion of the respective immunodominant epitopes (IDEs) in mutant virus Δ4IDE revealed a still efficient control of infection. In a murine model of hematopoietic cell transplantation and infection with Δ4IDE, an mCMV-specific open reading frame (ORF) library screening assay indicated a strong CD8 T cell reactivity against the ORF-M54 product, the highly conserved and essential mCMV homolog of human CMV DNA polymerase UL54, which is a known inducer of in vivo protection against mCMV by DNA immunization. Applying bioinformatic algorithms for CD8 T cell epitope prediction, the top-scoring peptides were used to stimulate ex vivo-isolated CD8 T cells and to generate cytolytic T cell lines; yet, this approach failed to identify M54 epitope(s). As an alternative, a peptide library consisting of 549 10-mers with an offset of two amino acids (aa), covering the complete aa-sequence of the M54 protein, was synthesized and used for the stimulation. A region of 12 aa proved to encompass an epitope. An 'alanine walk' over this antigenic 12-mer and all possible 11-, 10- and 9-mers derived thereof revealed aa-residues critical for antigenicity, and terminal truncations identified the H-2D(d) presented 8-mer M5483-90 as the optimal epitope. An increased frequency of the corresponding CD8 T cells in the absence of the 4 IDEs indicated immunodomination by the IDE-specific CD8 T cells as a mechanism by which the generation of M54-specific CD8 T cells is inhibited after infection with wild-type mCMV.

Immune control in the absence of immunodominant epitopes: implications for immunotherapy of cytomegalovirus infection with antiviral CD8 T cells.

Adoptive transfer of virus-specific donor-derived CD8 T cells is a therapeutic option to prevent cytomegalovirus (CMV) disease in recipients of hematopoietic cell transplantation. Due to their high coding capacity, human as well as animal CMVs have the potential to encode numerous CD8 T cell epitopes. Although the CD8 T cell response to CMVs is indeed broadly specific in that it involves epitopes derived from almost every open reading frame when tested for cohorts of immune CMV carriers representing the polymorphic MHC/HLA distribution in the population, the response in any one individual is directed against relatively few epitopes selected by the private combination of MHC/HLA alleles. Of this individually selected set of epitopes, few epitopes are 'immunodominant' in terms of magnitude of the response directed against them, while others are 'subdominant' according to this definition. In the assumption that 'immunodominance' indicates 'relevance' in antiviral control, research interest was focused on the immunodominant epitopes (IDEs) and their potential use in immunotherapy and in vaccines. The murine model has provided 'proof of concept' for the efficacy of CD8 T cell therapy of CMV infection. By experimental modulation of the CD8 T cell 'immunome' of murine CMV constructing an IDE deletion mutant, we have used this established cytoimmunotherapy model (a) for evaluating the actual contribution of IDEs to the control of infection and (b) for answering the question whether antigenicity-determining codon polymorphisms in IDE-encoding genes of CMV strains impact on the efficacy of CD8 T cell immunotherapy in case the donor and the recipient harbor different CMV strains.

Antigen presentation under the influence of 'immune evasion' proteins and its modulation by interferon-gamma: implications for immunotherapy of cytomegalovirus infection with antiviral CD8 T cells.

Cytomegalovirus (CMV) disease with multiple organ manifestations is the most feared viral complication limiting the success of hematopoietic cell transplantation as a therapy of hematopoietic malignancies. A timely endogenous reconstitution of CD8 T cells controls CMV infection, and adoptive transfer of antiviral CD8 T cells is a therapeutic option to prevent CMV disease by bridging the gap between an early CMV reactivation and delayed endogenous reconstitution of protective immunity. Preclinical research in murine models has provided 'proof of concept' for CD8 T-cell therapy of CMV disease. Protection by CD8 T cells appears to be in conflict with the finding that CMVs encode proteins that inhibit antigen presentation to CD8 T cells by interfering with the constitutive trafficking of peptide-loaded MHC class I molecules (pMHC-I complexes) to the cell surface. Here, we have systematically explored antigen presentation in the presence of the three currently noted immune evasion proteins of murine CMV in all possible combinations and its modulation by pre-treatment of cells with interferon-gamma (IFN-γ). The data reveal improvement in antigen processing by pre-treatment with IFN-γ can almost overrule the inhibitory function of immune evasion molecules in terms of pMHC-I expression levels capable of triggering most of the specific CD8 T cells, though the intensity of stimulation did not retrieve their full functional capacity. Notably, an in vivo conditioning of host tissue cells with IFN-γ in adoptive cell transfer recipients constitutively overexpressing IFN-γ (B6-SAP-IFN-γ mice) enhanced the antiviral efficiency of CD8 T cells in this transgenic cytoimmunotherapy model.

Parameters determining the efficacy of adoptive CD8 T-cell therapy of cytomegalovirus infection.

Reactivation of latent cytomegalovirus (CMV) in the transient state of immunodeficiency after hematopoietic cell transplantation (HCT) is the most frequent and severe viral complication endangering leukemia therapy success. By infecting the bone marrow (BM) stroma of the transplantation recipient, CMV can directly interfere with BM repopulation by the transplanted donor-derived hematopoietic cells and thus delay immune reconstitution of the recipient. Cytopathogenic virus spread in tissues can result in CMV disease with multiple organ manifestations of which interstitial pneumonia is the most feared. There exists a 'window of risk' between hematoablative treatment and reconstitution of antiviral immunity after HCT, whereby timely reconstitution of antiviral CD8 T cells is a recognized positive prognostic parameter for the control of reactivated CMV infection and prevention of CMV disease. Supplementation of endogenous reconstitution by adoptive cell transfer of 'ready-to-go' effector and/or memory virus epitope-specific CD8 T cells is a therapeutic option to bridge the 'window of risk.' Preclinical research in murine models of CMV disease has been pivotal by providing 'proof of concept' for a benefit from CD8 T-cell therapy of HCT-associated CMV disease (reviewed in Holtappels et al. Med Microbiol Immunol 197:125-134, 2008). Here, we give an update of our previous review with focus on parameters that determine the efficacy of adoptive immunotherapy of CMV infection by antiviral CD8 T cells in the murine model.

Antigen-presenting cells of haematopoietic origin prime cytomegalovirus-specific CD8 T-cells but are not sufficient for driving memory inflation during viral latency.

Expansion of the CD8 T-cell memory pool, also known as 'memory inflation', for certain but not all viral epitopes in latently infected host tissues is a special feature of the immune response to cytomegalovirus. The L(d)-presented murine cytomegalovirus (mCMV) immediate-early (IE) 1 peptide is the prototype of an epitope that is associated with memory inflation. Based on the detection of IE1 transcripts in latently infected lungs it was previously proposed that episodes of viral gene expression and antigenic activity due to desilencing of a limited number of viral genes may drive epitope-specific memory inflation. This would imply direct antigen presentation through latently infected host tissue cells rather than cell death-associated cross-presentation of viral antigens derived from productively infected cells through uninfected, professional antigen-presenting cells (profAPCs). To address the role of bone marrow-derived profAPCs in CD8 T-cell priming and memory to mCMV, we have used here a combined sex-mismatched and MHC class-I mismatched dual-marker bone marrow chimera model in which presentation of the IE1 epitope is restricted to donor-derived sry(+)L(d+) cells of haematopoietic differentiation lineages. Successful CD8 T-cell priming specific for the L(d)- and D(d)-presented inflationary epitopes IE1 and m164, respectively, but selective failure in IE1 epitope-specific memory inflation in these chimeras indicates different modes of antigen presentation involved in CD8 T-cell priming and memory inflation. These data suggest that memory inflation during mCMV latency requires expression of the epitope-presenting MHC class-I molecule by latently infected non-haematopoietic host tissue cells and thus predicts a role for direct antigen presentation in memory inflation.

Cytomegalovirus misleads its host by priming of CD8 T cells specific for an epitope not presented in infected tissues.

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 Db-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.

The efficacy of antigen processing is critical for protection against cytomegalovirus disease in the presence of viral immune evasion proteins.

Cytomegaloviruses (CMVs) code for immunoevasins, glycoproteins that are specifically dedicated to interfere with the presentation of antigenic peptides to CD8 T cells. Nonetheless, the biological outcome is not an immune evasion of the virus, since CD8 T cells can control CMV infection even when immunoevasins are expressed. Here, we compare the processing of a protective and a nonprotective epitope derived from the same viral protein, the antiapoptotic protein M45 in the murine model. The data provide evidence to conclude that protection against CMVs critically depends on antigenic peptides generated in an amount sufficient to exhaust the inhibitory capacity of immunoevasins.

Immune evasion proteins enhance cytomegalovirus latency in the lungs.

CD8 T cells control cytomegalovirus (CMV) infection in bone marrow transplantation recipients and persist in latently infected lungs as effector memory cells for continuous sensing of reactivated viral gene expression. Here we have addressed the question of whether viral immunoevasins, glycoproteins that specifically interfere with antigen presentation to CD8 T cells, have an impact on viral latency in the murine model. The data show that deletion of immunoevasin genes in murine CMV accelerates the clearance of productive infection during hematopoietic reconstitution and leads to a reduced latent viral genome load, reduced latency-associated viral transcription, and a lower incidence of recurrence in lung explants.

Subdominant CD8 T-cell epitopes account for protection against cytomegalovirus independent of immunodomination.

Cytomegalovirus (CMV) infection continues to be a complication in recipients of hematopoietic stem cell transplantation (HSCT). Preexisting donor immunity is recognized as a favorable prognostic factor for the reconstitution of protective antiviral immunity mediated primarily by CD8 T cells. Furthermore, adoptive transfer of CMV-specific memory CD8 T (CD8-T(M)) cells is a therapeutic option for preventing CMV disease in HSCT recipients. Given the different CMV infection histories of donor and recipient, a problem may arise from an antigenic mismatch between the CMV variant that has primed donor immunity and the CMV variant acquired by the recipient. Here, we have used the BALB/c mouse model of CMV infection in the immunocompromised host to evaluate the importance of donor-recipient CMV matching in immundominant epitopes (IDEs). For this, we generated the murine CMV (mCMV) recombinant virus mCMV-DeltaIDE, in which the two memory repertoire IDEs, the IE1-derived peptide 168-YPHFMPTNL-176 presented by the major histocompatibility complex class I (MHC-I) molecule L(d) and the m164-derived peptide 257-AGPPRYSRI-265 presented by the MHC-I molecule D(d), are both functionally deleted. Upon adoptive transfer, polyclonal donor CD8-T(M) cells primed by mCMV-DeltaIDE and the corresponding revertant virus mCMV-revDeltaIDE controlled infection of immunocompromised recipients with comparable efficacy and regardless of whether or not IDEs were presented in the recipients. Importantly, CD8-T(M) cells primed under conditions of immunodomination by IDEs protected recipients in which IDEs were absent. This shows that protection does not depend on compensatory expansion of non-IDE-specific CD8-T(M) cells liberated from immunodomination by the deletion of IDEs. We conclude that protection is, rather, based on the collective antiviral potential of non-IDEs independent of the presence or absence of IDE-mediated immunodomination.

Noncanonical expression of a murine cytomegalovirus early protein CD8 T-cell epitope as an immediate early epitope based on transcription from an upstream gene.

Viral CD8 T-cell epitopes, represented by viral peptides bound to major histocompatibility complex class-I (MHC-I) glycoproteins, are often identified by "reverse immunology", a strategy not requiring biochemical and structural knowledge of the actual viral protein from which they are derived by antigen processing. Instead, bioinformatic algorithms predicting the probability of C-terminal cleavage in the proteasome, as well as binding affinity to the presenting MHC-I molecules, are applied to amino acid sequences deduced from predicted open reading frames (ORFs) based on the genomic sequence. If the protein corresponding to an antigenic ORF is known, it is usually inferred that the kinetic class of the protein also defines the phase in the viral replicative cycle during which the respective antigenic peptide is presented for recognition by CD8 T cells. We have previously identified a nonapeptide from the predicted ORFm164 of murine cytomegalovirus that is presented by the MHC-I allomorph H-2 Dd and that is immunodominant in BALB/c (H-2d haplotype) mice. Surprisingly, although the ORFm164 protein gp36.5 is expressed as an Early (E) phase protein, the m164 epitope is presented already during the Immediate Early (IE) phase, based on the expression of an upstream mRNA starting within ORFm167 and encompassing ORFm164.

Optimized recombinant dense bodies of human cytomegalovirus efficiently prime virus specific lymphocytes and neutralizing antibodies without the addition of adjuvant.

Control of human cytomegalovirus (HCMV) infection correlates with the reconstitution of antiviral T lymphocytes in haematopoietic stem cell transplant recipients. A vaccine to foster this reconstitution and to ameliorate the severe consequences of HCMV reactivation is yet unavailable. This work focused on providing a rationale for the amendment of the yields and the antigenic composition of a vaccine, based on subviral dense bodies (DB) of HCMV. Modified DB were generated that contained the HLA-A2 presented IE1 model peptide TMYGGISLL, integrated at different positions in the major DB protein pp65. Insertion at position W175 of pp65 allowed efficient formation of recDB in the cytoplasm of infected cells and resulted in considerable yields of these particles. Even in the absence of adjuvant, these particles proved to be highly immunogenic with respect to CD8 and CD4 T cell and neutralizing antibody responses.

Adoptive CD8 T cell control of pathogens cannot be improved by combining protective epitope specificities.

Adoptive transfer of CD8 T cells has the potential to cure infectious or malignant diseases that are refractory to conventional chemotherapy. A practically important but still unanswered question is whether mixtures of protective CD8 T cells with different epitope specificities mediate more efficient effector cell functions than do the monospecific individual CD8 T cell populations. In this study, we have addressed this issue for models of viral and bacterial infection. CD8 T cell-mediated cytotoxicity in vitro and protection in vivo were assessed to test whether CD8 T cell lines cooperate in target cell lysis and control of infection, respectively. Our data clearly show that mixtures of cytolytic T cell lines specific for different epitopes of either murine cytomegalovirus or Listeria monocytogenes do not act synergistically. An efficient anti-infectious protection thus proved to be dependent primarily on the number of transferred protective CD8 T cells rather than on the cooperative effects of multiple specificities.

Epitope-specific in vivo protection against cytomegalovirus disease by CD8 T cells in the murine model of preemptive immunotherapy.

Preclinical research in murine models as well as subsequent clinical trials have concordantly revealed a high protective potential of antiviral CD8 T cells, of donor-derived 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.

Cytomegalovirus encodes a positive regulator of antigen presentation.

Murine cytomegalovirus encodes three regulators of antigen presentation to antiviral CD8 T cells. According to current paradigms, all three regulators are committed to the inhibition of the presentation of antigenic peptides. Whereas m152/gp40 catalyzes the retention of peptide-loaded major histocompatibility complex (MHC) class I molecules in a cis-Golgi compartment, m06/gp48 binds stably to class I molecules and directs them into the cellular cargo-sorting pathway of lysosomal degradation. Regulator m04/gp34 also binds stably to class I molecules, but unlike m152 and m06, it does not downmodulate MHC class I cell surface expression. It has entered the literature as a direct inhibitor of T-cell recognition of the MHC-peptide complex at the cell surface. In this work, we have studied the presentation of antigenic viral peptides in cells infected with a comprehensive set of mutant viruses expressing the three regulators separately as well as in all possible combinations. The results redefine m04 as a positive regulator dedicated to the facilitation of antigen presentation. When expressed alone, it did not inhibit T-cell recognition, and when expressed in the presence of m152, it restored antigen presentation by antagonizing the inhibitory function of m152. Its intrinsic positive function, however, was antagonized and even slightly overcompensated for by the negative regulator m06. In an adoptive cell transfer model, the opposing forces of the three regulators were found to govern immune surveillance in the infected host. While negative regulators, also known as immunoevasins, are common, the existence of a positive regulator is without precedent and indicates an intriguing genetic potential of this virus to influence antigen presentation.

Highly protective in vivo function of cytomegalovirus IE1 epitope-specific memory CD8 T cells purified by T-cell receptor-based cell sorting.

Reconstitution of antiviral CD8 T cells is essential for controlling cytomegalovirus (CMV) infection after bone marrow transplantation. Accordingly, polyclonal CD8 T cells derived from BALB/c mice infected with murine CMV protect immunocompromised adoptive transfer recipients against CMV disease. The protective population comprises CD8 T cells with T-cell receptors (TCRs) specific for defined and for as-yet-unknown viral epitopes, as well as a majority of nonprotective cells with unrelated specificities. Defined epitopes include IE1/m123 and m164, which are immunodominant in terms of the magnitude of the CD8 T-cell response, and a panel of subordinate epitopes (m04, m18, M45, M83, and M84). While cytolytic T-lymphocyte lines (CTLLs) were shown to be protective regardless of the immunodominance of the respective epitope, the individual contributions of in vivo resident epitope-specific CD8 T cells to the antiviral control awaited investigation. The IE1 peptide 168-YPHFMPTNL-176 is generated from the immediate-early protein 1 (IE1) (pp89/76) of murine CMV and is presented by the major histocompatibility complex class I (MHC-I) molecule Ld. To quantitate its contribution to the protective potential of a CD8-T memory (CD8-TM) cell population, IE1-TCR+ and IE1-TCR- CD8-TM cells were purified by epitope-specific cell sorting with IE1 peptide-loaded MHC-immunoglobulin G1 dimers as ligands of cognate TCRs. Of relevance for clinical approaches to an adoptive cellular immunotherapy, sorted IE1 epitope-specific CD8-TM cells were found to be exceedingly protective upon adoptive transfer. Compared with CTLLs specific for the same epitope and of comparable avidity and TCR beta-chain variable region (Vbeta)-defined polyclonality, sorted CD8-TM cells proved to be superior by more than 2 orders of magnitude.

Identification of a K(d)-restricted antigenic peptide encoded by murine cytomegalovirus early gene M84.

The two sister cytomegaloviruses (CMVs), human and murine CMV, have both evolved immune evasion functions that interfere with the major histocompatibility complex class I (MHC-I) pathway of antigen processing and presentation and are effectual in the early (E) phase of virus gene expression. However, studies on murine CMV have shown that E-phase immune evasion is leaky. An E-phase protein involved in immune evasion, namely m04-gp34, was found to simultaneously account for an antigenic peptide presented by the MHC-I molecule D(d). Recent work has demonstrated the induction of protective immunity specific for the E-phase protein M84-p65, one of two murine CMV homologues of the human CMV matrix protein UL83-pp65. In this study, the identification of the MHC-I K(d)-restricted M84 peptide (297)AYAGLFTPL(305) is documented. This peptide is the third antigenic peptide described for murine CMV and the second that escapes immunosubversive mechanisms.

Two antigenic peptides from genes m123 and m164 of murine cytomegalovirus quantitatively dominate CD8 T-cell memory in the H-2d haplotype.

The importance of CD8 T cells for the control of cytomegalovirus (CMV) infection has raised interest in the identification of immunogenic viral proteins as candidates for vaccination and cytoimmunotherapy. The final aim is to determine the viral "immunome" for any major histocompatibility complex class I molecule by antigenicity screening of proteome-derived peptides. For human CMV, there is a limitation to this approach: the T cells used as responder cells for peptide screening are usually memory cells that have undergone in vivo selection. On this basis, pUL83 (pp65) and pUL123 (IE1 or pp68 to -72) were classified as immunodominant proteins. It is an open question whether this limited "memory immunome" really reflects the immunogenic potential of the human CMV proteome. Here we document an analogous focus of the memory repertoire on two proteins of murine CMV. Specifically, ca. 80% of all memory CD8 T cells in the spleen as well as in persisting pulmonary infiltrates were found to be specific for the known IE1 peptide 168YPHFMPTNL176 and for the peptide 257AGPPRYSRI265, newly defined here, derived from open reading frame m164. Notably, CD8 T-cell lines of both specificities protected against acute infection upon adoptive transfer. In contrast, the natural immune response to acute infection in draining lymph nodes and in the lungs indicated a somewhat broader specificity repertoire. We conclude that the low number of antigenic peptides identified so far for CMVs reflects a focused memory repertoire, and we predict that more antigenic peptides will be disclosed by analysis of the acute immune response.

Early gene m18, a novel player in the immune response to murine cytomegalovirus.

The identification of all antigenic peptides encoded by a pathogen, its T cell 'immunome', is a research aim for rational vaccine design. Screening of proteome-spanning peptide libraries or computational prediction is used to identify antigenic peptides recognized by CD8 T cells. Based on their high coding capacity, cytomegaloviruses (CMVs) could specify numerous antigenic peptides. Yet, current evidence indicates that the memory CD8 T cell response in a given haplotype is actually focused on a few viral proteins. CMVs actively interfere with antigen processing and presentation by the expression of immune evasion proteins. In the case of murine CMV (mCMV), these proteins are effectual in the early (E) phase of the virus replication cycle and should thus preclude the presentation of peptides derived from E proteins. Notably, the m18 gene is here added to a growing list of mCMV E genes that encode antigenic peptides in spite of the E phase immune evasion strategies of the virus.

Processing and presentation of murine cytomegalovirus pORFm164-derived peptide in fibroblasts in the face of all viral immunosubversive early gene functions.

CD8 T cells are the principal effector cells in the resolution of acute murine cytomegalovirus (mCMV) infection in host organs. This undoubted antiviral and protective in vivo function of CD8 T cells appeared to be inconsistent with immunosubversive strategies of the virus effected by early (E)-phase genes m04, m06, and m152. The so-called immune evasion proteins gp34, gp48, and gp37/40, respectively, were found to interfere with peptide presentation at different steps in the major histocompatibility complex (MHC) class I pathway of antigen processing and presentation in fibroblasts. Accordingly, they were proposed to prevent recognition and lysis of infected fibroblasts by cytolytic T lymphocytes (CTL) during the E phase of viral gene expression. We document here that the previously identified MHC class I D(d)-restricted antigenic peptide (257)AGPPRYSRI(265) encoded by gene m164 is processed as well as presented for recognition by m164-specific CTL during the E and late phases of viral replication in the very same cells in which the immunosubversive viral proteins are effectual in preventing the presentation of processed immediate-early 1 (m123-exon 4) peptide (168)YPHFMPTNL(176). Thus, while immunosubversion is a reality, these mechanisms are apparently not as efficient as the term immune evasion implies. The pORFm164-derived peptide is the first noted peptide that constitutively escapes the immunosubversive viral functions. The most important consequence is that even the concerted action of all immunosubversive E-phase proteins eventually fails to prevent immune recognition in the E phase. The bottom-line message is that there exists no immune evasion of mCMV in fibroblasts.