Jürgen Podlech

Major Histocompatibility Complex Class I Allele-specific Cooperative and Competitive Interactions between Immune Evasion Proteins of Cytomegalovirus

Cytomegaloviruses (CMVs) deploy a set of genes for interference with antigen presentation in the major histocompatibility complex (MHC) class I pathway. In murine CMV (MCMV), three genes were identified so far: m04/gp34, m06/gp48, and m152/gp40. While their function as immunoevasins was originally defined after their selective expression, this may not necessarily reflect their biological role during infection. The three immunoevasins might act synergistically, but they might also compete for their common substrate, the MHC class I complexes. To approach this question in a systematic manner, we have generated a complete set of mutant viruses with deletions of the three genes in all seven possible combinations. Surface expression of a set of MHC class I molecules specified by haplotypes H-2d (Kd, Dd, and Ld) and H-2b (Kb and Db) was the parameter for evaluation of the interference with class I trafficking. The data show the following: first, there exists no additional MCMV gene of major influence on MHC class I surface expression; second, the strength of the inhibitory effect of immunoevasins shows an allele-specific hierarchy; and third, the immunoevasins act not only synergistically but can, in certain combinations, interact antagonistically. In essence, this work highlights the importance of studying the immunosubversive mechanisms of cytomegaloviruses in the context of gene expression during the viral replicative cycle in infected cells.

CD8 T Cells Control Cytomegalovirus Latency by Epitope-Specific Sensing of Transcriptional Reactivation

ABSTRACT During murine cytomegalovirus (mCMV) latency in the lungs, most of the viral genomes are transcriptionally silent at the major immediate-early locus, but rare and stochastic episodes of desilencing lead to the expression of IE1 transcripts. This low-frequency but perpetual expression is accompanied by an activation of lung-resident effector-memory CD8 T cells specific for the antigenic peptide 168-YPHFMPTNL-176, which is derivedfrom the IE1 protein. These molecular and immunological findings were combined in the “silencing/desilencing and immune sensing hypothesis” of cytomegalovirus latency and reactivation. This hypothesis proposes that IE1 gene expression proceeds to cell surface presentation of the IE1 peptide by the major histocompatibility complex (MHC) class I molecule Ld and that its recognition by CD8 T cells terminates virus reactivation. Here we provide experimental evidence in support of this hypothesis. We generated mutant virus mCMV-IE1-L176A, in which the antigenic IE1 peptide is functionally deleted by a point mutation of the C-terminal MHC class I anchor residue Leu into Ala. Two revertant viruses, mCMV-IE1-A176L and the wobble nucleotide-marked mCMV-IE1-A176L*, in which Leu is restored by back-mutation of Ala codon GCA into Leu codons CTA and CTT, respectively, were constructed. Pulmonary latency of the mutant virus was found to be associated with an increased prevalence of IE1 transcription and with events of IE3 transactivator splicing. In conclusion, IE1-specific CD8 T cells recognize and terminate virus reactivation in vivo at the first opportunity in the reactivated gene expression program. The perpetual gene expression and antigen presentation might represent the driving molecular force in CMV-associated immunosenescence.

Mouse models of cytomegalovirus latency: overview

BACKGROUND The molecular regulation of viral latency and reactivation is a central unsolved issue in the understanding of cytomegalovirus (CMV) biology. Like human CMV (hCMV), murine CMV (mCMV) can establish a latent infection in cells of the myeloid lineage. Since mCMV genome remains present in various organs after its clearance from hematopoietic cells first in bone marrow and much later in blood, there must exist one or more widely distributed cell type(s) representing the cellular site(s) of enduring mCMV latency in host tissues. Endothelial cells and histiocytes are candidates, but the question is not yet settled. Another long debated problem appears to be solved: mCMV establishes true molecular latency rather than a low-level persistence of productive infection. This conclusion is based on two recent advances. First, on a highly improved assay of infectivity, and second, on very sensitive RT PCRs for detecting viral transcripts during latency. In essence, infectious virus and productive cycle transcripts, such as transcripts of early-phase gene M55 (gB) and ie3 transcripts specifying the essential transactivator protein IE3, were found to be absent during mCMV latency in the lungs. OBJECTIVES We will here review recent data on the variegated expression of IE-phase genes ie1 and ie2 during mCMV latency in the lungs, and on the expression patterns found in transcriptional foci during induced reactivation. We will discuss immunological implications of ie1 gene expression during latency and will speculate a bit on how CD8 T cells might trigger latency-associated ie1 gene expression in a regulatory circuit.

Frequent Coinfection of Cells Explains Functional In Vivo Complementation between Cytomegalovirus Variants in the Multiply Infected Host

ABSTRACT In contrast to many other virus infections, primary cytomegalovirus (CMV) infection does not fully protect against reinfection. Accordingly, clinical data have revealed a coexistence of multiple human CMV variants/strains in individual patients. Notably, the phenomenon of multiple infection was found to correlate with increased virus load and severity of CMV disease. Although of obvious medical relevance, the mechanism underlying this correlation is unknown. A weak immune response in an individual could be responsible for a more severe disease and for multiple infections. Alternatively, synergistic contributions of variants that differ in their biological properties can lead to qualitative changes in viral fitness by direct interactions such as genetic recombination or functional complementation within coinfected host cells. We have addressed this important question paradigmatically with the murine model by differently designed combinations of two viruses employed for experimental coinfection of mice. Specifically, a murine cytomegalovirus (MCMV) mutant expressing Cre recombinase was combined for coinfection with a mutant carrying Cre-inducible green fluorescent protein gene, and attenuated mutants were combined for coinfection with wild-type virus followed by two-color in situ hybridization studies visualizing the replication of the two viruses in infected host organs. These different approaches concurred in the conclusion that coinfection of host cells is more frequent than statistically predicted and that this coinfection alters virus fitness by functional trans-complementation rather than by genetic recombination. The reported findings make a major contribution to our molecular understanding of enhanced CMV pathogenicity in the multiply infected host.

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

ABSTRACT 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.

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.

Murine Model of Interstitial Cytomegalovirus Pneumonia in Syngeneic Bone Marrow Transplantation: Persistence of Protective Pulmonary CD8-T-Cell Infiltrates after Clearance of Acute Infection

ABSTRACT Interstitial pneumonia (IP) is a severe organ manifestation of cytomegalovirus (CMV) disease in the immunocompromised host, in particular in recipients of bone marrow transplantation (BMT). Diagnostic criteria for the definition of CMV-IP include clinical evidence of pneumonia together with CMV detected in bronchoalveolar lavage or lung biopsy. We have used the model of syngeneic BMT and simultaneous infection of BALB/c mice with murine CMV for studying the pathogenesis of CMV-IP by controlled longitudinal analysis. A disseminated cytopathic infection of the lungs with fatal outcome was observed only when reconstituting CD8 T cells were depleted. Neither CD8 nor CD4 T cells mediated an immunopathogenesis of acute CMV-IP. By contrast, after efficient hematolymphopoietic reconstitution, viral replication in the lungs was moderate and focal. The histopathological picture was dominated by preferential infiltration of CD8 T cells confining viral replication to inflammatory foci. Notably, after clearance of acute infection, CD62Llo and CD62Lhi subsets of CD44+ 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.

Highly Protective In Vivo Function of Cytomegalovirus IE1 Epitope-Specific Memory CD8 T Cells Purified by T-Cell Receptor-Based Cell Sorting

ABSTRACT 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 β-chain variable region (Vβ)-defined polyclonality, sorted CD8-TM cells proved to be superior by more than 2 orders of magnitude.

The major virus-producing cell type during murine cytomegalovirus infection, the hepatocyte, is not the source of virus dissemination in the host.

The course of systemic viral infections is determined by the virus productivity of infected cell types and the efficiency of virus dissemination throughout the host. Here, we used a cell-type-specific virus labeling system to quantitatively track virus progeny during murine cytomegalovirus infection. We infected mice that expressed Cre recombinase selectively in vascular endothelial cells or hepatocytes with a murine cytomegalovirus for which Cre-mediated recombination would generate a fluorescently labeled virus. We showed that endothelial cells and hepatocytes produced virus after direct infection. However, in the liver, the main contributor to viral load in the mouse, most viruses were produced by directly infected hepatocytes. Remarkably, although virus produced in hepatocytes spread to hepatic endothelial cells (and vice versa), there was no significant spread from the liver to other organs. Thus, the cell type producing the most viruses was not necessarily the one responsible for virus dissemination within the host.

The Putative Natural Killer Decoy Early Gene m04 (gp34) of Murine Cytomegalovirus Encodes an Antigenic Peptide Recognized by Protective Antiviral CD8 T Cells

ABSTRACT Several early genes of murine cytomegalovirus (MCMV) encode proteins that mediate immune evasion by interference with the major histocompatibility complex class I (MHC-I) pathway of antigen presentation to cytolytic T lymphocytes (CTL). Specifically, the m152 gene product gp37/40 causes retention of MHC-I molecules in the endoplasmic reticulum (ER)-Golgi intermediate compartment. Lack of MHC-I on the cell surface should activate natural killer (NK) cells recognizing the “missing self.” The retention, however, is counteracted by the m04early gene product gp34, which binds to folded MHC-I molecules in the ER and directs the complex to the cell surface. It was thus speculated that gp34 might serve to silence NK cells and thereby complete the immune evasion of MCMV. In light of these current views, we provide here results demonstrating an in vivo role for gp34 in protective antiviral immunity. We have identified an antigenic nonapeptide derived from gp34 and presented by the MHC-I molecule Dd. Besides the immunodominant immediate-early nonapeptide consisting of IE1 amino acids 168-176 (IE1168-176), the early nonapeptide m04243-251 is the second antigenic peptide described for MCMV. The primary immune response to MCMV generates significant m04-specific CD8 T-cell memory. Upon adoptive transfer into immunodeficient recipients, an m04-specific CTL line controls MCMV infection with an efficacy comparable to that of an IE1-specific CTL line. Thus, gp34 is the first noted early protein of MCMV that escapes viral immune evasion mechanisms. These data document that MCMV is held in check by a redundance of protective CD8 T cells recognizing antigenic peptides in different phases of viral gene expression.