The Memory Cytotoxic T-Lymphocyte (CTL) Response to Human Cytomegalovirus Infection Contains Individual Peptide-Specific CTL Clones That Have Undergone Extensive Expansion In Vivo

Human cytomegalovirus (HCMV)-specific CD8⁺ cytotoxic T lymphocytes (CTL) appear to play an important role in the control of virus replication and in protection against HCMV-related disease. We have previously reported high frequencies of memory CTL precursors (CTLp) specific to the HCMV tegument protein pp65 in the peripheral blood of healthy virus carriers. In some individuals, the CTL response to this protein is focused on only a single epitope, whereas in other virus carriers CTL recognized multiple epitopes which we identified by using synthetic peptides. We have analyzed the clonal composition of the memory CTL response to four of these pp65 epitopes by sequencing the T-cell receptors (TCR) of multiple independently derived epitope-specific CTL clones, which were derived by formal single-cell cloning or from clonal CTL microcultures. In all cases, we have observed a high degree of clonal focusing: the majority of CTL clones specific to a defined pp65 peptide from any one virus carrier use only one or two different TCRs at the level of the nucleotide sequence. Among virus carriers who have the same major histocompatibility complex (MHC) class I allele, we observed that CTL from different donors that recognize the same peptide-MHC complex often used the same Vβ segment, although other TCR gene segments and CDR3 length were not in general conserved. We have also examined the clonal composition of CTL specific to pp65 peptides in asymptomatic human immunodeficiency virus-infected individuals. We have observed a similarly focused peptide-specific CTL response. Thus, the large population of circulating HCMV peptide-specific memory CTLp in virus carriers in fact contains individual CTL clones that have undergone extensive clonal expansion in vivo.

CD8⁺ cytotoxic T lymphocytes (CTL) recognize virus-infected cells via the T-cell receptor (TCR), an αβ heterodimer that has specificity for the peptide antigen presented by major histocompatibility complex (MHC) class I molecules. During T-cell development in the thymus, the TCR β-chain is constructed by rearrangement of variable (V), diversity (D), and joining (J) gene segments, and the α-chain by rearrangement of V and J segments. Additional diversity is generated by imperfect joining of these segments, exonucleotide nibbling at the joins, and addition of non-germ line-encoded N-region nucleotides (25). The regions spanning the V-D-J and V-J joins constitute the hypervariable CDR3 regions which are thought to interact with the middle of the bound peptide and to account for approximately 50% of the TCR’s interaction with peptide (14, 15, 20). The α- and β-chain complementarity determining regions CDR1, which reside within the TCR V segments, are thought to interact with the N and C termini of a peptide that is bound to MHC. By contrast, Vα and Vβ CDR2s are thought to interact predominantly with the MHC itself (14, 15).Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that infects between 60 and 90% of individuals, depending on the population studied. After primary HCMV infection, the virus persists lifelong in a latent state in cells of the myeloid lineage and under the control of the immune system (5). HCMV reactivation can, however, cause serious disease in immunocompromised individuals, such as patients with advanced human immunodeficiency virus (HIV) infection (30) and patients who have undergone bone marrow transplantation (33). Evidence from animal models (32) and from studies of immunosuppressed humans (39) indicates that virus-specific CD8⁺ CTL have a role in protection against CMV disease.We previously studied in detail the HCMV-specific CTL response in healthy virus carriers. All seropositive donors had high frequencies of MHC-restricted HCMV-specific memory CTL precursors in peripheral blood and strongly recognized one of the viral tegument proteins, pp65. In some donors, the CTL response to this protein was highly focused, recognizing only a single epitope within pp65, whereas in others the CTL recognized multiple pp65 peptides (41 and unpublished data).The aim of this study was to examine the clonal composition of the memory CTL response to HCMV pp65 by determining how many different CTL clones are involved in the recognition of a given pp65 peptide. In order to do this, we analyzed the TCR α- and β-chain usage of multiple independently derived peptide-specific CTL clones from healthy virus carriers.Previous studies have examined the heterogeneity of the CTL response to other human virus infections within single subjects (2, 8, 11, 18, 19, 22, 38) or between different donors (2, 6, 8, 11, 23, 38). In the most extreme cases, a very high degree of TCR focusing has been seen: in a study of one HIV-positive individual’s CTL response to an HLA-B14-restricted HIV env peptide, the same TCR was used by 9 of 10 peptide-specific CTL clones, each derived at different time points over the course of 36 months (22). Similarly, multiple independent CTL clones specific to an HLA-B8-restricted Epstein-Barr virus (EBV) peptide derived from one virus carrier at one time point all used the same TCR (2). The CTL response to different human T-lymphotropic virus type 1 (HTLV-1) peptides has been observed to be oligoclonal within individual donors (38). However, in a variety of other human and mouse viral infections within a given individual, the repertoire of CTL specific for a given peptide has been highly heterogeneous (8, 11, 18, 19).The TCRs of CTL obtained from different donors that recognize the same peptide-MHC complex often show some conservation of gene segment usage, although they differ in hypervariable sequence. For example, Vβ segments and certain β-chain CDR3 motifs were conserved between TCR that recognized an HLA-A2-restricted influenza virus peptide in CTL clones derived from different donors (23); the same phenomenon has been seen for an HLA-B27 restricted influenza virus peptide (6) and an HLA-A11-restricted EBV peptide (8). A much higher degree of TCR conservation has also been seen; the same TCR α- and β-chain protein sequences were used by CTL clones from four of five unrelated donors that recognized an HLA-B8 restricted EBV peptide (2). In the case of HTLV-1, CTL from different donors that were specific to the same peptide used largely unrelated TCR (38).For all of the human viruses so far studied, the clonal composition of virus-specific CTL has only been examined for a very few viral peptide-MHC combinations, sometimes in only one donor or at only one time point. In this study, we have therefore examined multiple CTL clones specific to a total of four pp65 peptides, all restricted by three different HLA alleles. We have derived these clones from six healthy virus carriers at one to four time points up to 18 months apart. To identify CTL clonotypes for longitudinal studies and to determine whether HIV infection modifies the clonal composition of HCMV-specific CTL, we have also examined pp65-specific memory CTL in two asymptomatic HIV-infected subjects who are HCMV seropositive. For any given individual, whether HIV seropositive or seronegative, our results indicate that the memory CTL response to individual HCMV pp65 epitopes is highly focused and contains CTL clones that have undergone extensive expansion in vivo.