Although cytomegalovirus (CMV) expresses proteins that interfere with antigen presentation by class I major histocompatibility complex (MHC) molecules, CD8 ؉ cytotoxic T cells (CTLs) are indispensable for controlling infection and maintaining latency. Here, a cytokine flow cytometry assay that employs fibroblasts infected with a mutant strain of CMV (RV798), which is deleted of the 4 viral genes that are responsible for interfering with class I MHC presentation, was used to examine the frequency and specificity of the CD8 ؉ CTLs to CMV in immunocompetent CMV-seropositive individuals. A large fraction of the CD8 ؉ CTL response was found to be specific for viral antigens expressed during the immediate early and early phases of virus replication and presented by fibroblasts infected with RV798 but not wild-type CMV. These results demonstrate that the inhibition of class I antigen presentation observed in CMV-infected cells in vitro is not sufficient to prevent the induction of a broad repertoire of CD8 ؉ CTLs after natural infection in vivo. Thus, reconstitution of T-cell immunity in immunodeficient patients by cell therapy or by vaccination may need to target multiple viral antigens to completely restore immunologic control of

Human cytomegalovirus (CMV) may cause severe disease in immunosuppressed hosts including hematopoietic stem cell transplant recipients. The components of host immunity that provide protection from progressive infection have not been completely defined. Studies in immunocompromised CMV-infected humans, and in mice infected with murine cytomegalovirus (mCMV), have shown that recovery of class I major histocompatibility complex (MHC) restricted CD8+ cytotoxic T lymphocytes (CTL) that recognize viral peptides play a key role in containing viral replication. The importance of CTL specific for individual antigens in immune control of CMV has been difficult to determine because cells permissively infected with human CMV express over 160 viral proteins during the (IE), early (E), and late (L) phases of replication. Recent studies in immunocompetent humans have identified significant frequencies of CTL specific for multiple CMV antigens expressed at all stages of viral replication, despite the presence of viral proteins that interfere with class I antigen presentation. Thus, analysis of CMV-specific T cell immunity using one or a few peptides is likely to underestimate the magnitude and diversity of the host response. We have developed a genetic approach for characterizing CMV antigens that concurrently identifies the HLA restricting allele and enables rapid determination of the minimal epitope derived from any CMV gene. To evaluate this approach, PBMC from seropositive individuals were stimulated once in vitro with autologous fibroblasts infected with the RV798 strain of CMV, which lacks all class I immune evasion genes and enables display of all potentially antigenic peptides. The T cells were then screened against a panel of COS cells transfected with a plasmid library containing a majority of CMV ORFs and with each of the HLA alleles of the donor. Twenty-two CMV genes that were predominantly expressed at IE or E stages of infection were identified to encode antigens recognized by CTL from 4 normal donors. The median number of antigens recognized in each donor was 8 (range 4–12). Seventeen CMV peptides presented by a variety of common HLA class I molecules including HLA-C were subsequently mapped and two epitopes were found to be derived from alternative translation products or processing mechanisms. Memory T cells from other CMV seropositive individuals that shared the HLA restricting allele also recognized these novel epitopes. This genome scan was used successfully to identify the repertoire of CMV antigens recognized by CMV-specific CTL generated from CMV seropositive hematopoietic stem cell transplant donors and to determine which responses were transferred to the respective recipient post transplant. CTL specific for a broad repertoire of viral antigens comparable to that in the donor were found in some transplant recipients, while in others the response was dramatically restricted compared to the donor. These results further define the broad specificity of the CMV-specific CTL response in seropositive donors, enable comprehensive monitoring of the recovery of CMV-specific CTL in immunocompromised patients at risk for CMV disease, and may be useful for defining the specificity of CTL responses that correlate with control of virus replication.