Cytomegalovirus immune reconstitution occurs in recipients of allogeneic hematopoietic cell transplants irrespective of detectable cytomegalovirus infection.

The question of when immune reconstitution of cytomegalovirus (CMV)-specific CD8 T cells occurs after hematopoietic cell transplantation and, more specifically, to which CMV targets this immunity is likely to be directed remains poorly understood. The dependence of immune reconstitution on CMV reactivation is even less clear. To better understand these events, 44 CMV-seropositive HLA-A*0201 subjects were followed up at approximately days 40, 90, 120, 150, 180, and 360 after hematopoietic cell transplantation for CMV immunity as measured by 2 types of assays: (1) an HLA-A*0201 tetramer-binding assay for both CMV pp65 (pp65) and immediate-early 1 (IE-1) or (2) intracellular cytokine interferon gamma responses induced by pp65 or IE-1-derived peptides. To verify the reliability of IE-1-specific assays relative to the pp65-based assays, a pilot study first compared the development of IE-1-specific immunity in a subgroup by using multiple HLA-A*0201-restricted peptides, and then these recipients were followed up for 1 year for immunologic function and for CMV infection. The IE-1-specific response occurred to each of the 3 HLA-A*0201-restricted peptides studied (IE-1-256, -297, and -316), and there was no predominant IE peptide response. However, the immunodominant HLA-A*0201-restricted pp65 peptide was recognized significantly more frequently than these IE-1 peptides. When this was compared with the occurrence of CMV infection, the overall immune reactivity, as measured by the mean or median number of CD8+ T cells reactive to either pp65 or IE-1 peptides by intracellular cytokine or tetramer binding assay, was not significantly different in those with and without CMV infection. For patients who demonstrated reconstituted immunity to CMV at 1 year, all were reconstituted by 6 months, and the timing of the first observed immune reactivity to either of the pp65 or the IE peptides was not different in those with and without detectable CMV infection.

Characterization of host immunity to cytomegalovirus pp150 (UL32).

The basic phosphoprotein 150 (pp150), the product of UL32 (unique long domain 32) gene of human cytomegalovirus (CMV), is an abundant component of the viral tegument and a target of human leukocyte antigen (HLA)-restricted cytotoxic T cells (CTLs) after infection. Identification of minimal cytotoxic epitopes (MCEs) from this CMV protein is of importance for peptide-based vaccines and immunotherapeutic approaches. Several pp150-specific CTL clones were derived from peripheral blood mononuclear cells of healthy CMV-positive donors with autologous fibroblasts infected either with CMV AD169 or with a recombinant vaccinia virus expressing full-length pp150 protein. HLA A*0301- and HLA A*6801-restricted CD8+ pp150 T-cell clones derived from different donors were found to efficiently kill autologous CMV-infected fibroblasts. Fine mapping of each MCE first used a T-cell epitope prediction algorithm. Overlapping peptides within the recognized regions were screened. The analysis identified pp150(792-802) and pp150(945-955) as MCEs for the HLA A*6801 and the HLA A*0301 pp150 clones, respectively. In vitro stimulation by recombinant modified vaccinia Ankara virus expressing full-length pp150 elicited high frequencies of CMV-CTL and interferon gamma production specific for the MCE identified in all subjects. The consistent presence of pp150 T cells in CMV-exposed individuals supports a role for this antigen in shaping the antiviral CTL response and indicates that pp150 could be a pivotal constituent of prophylactic and therapeutic CMV vaccines.

Relative dominance of HLA-B*07 restricted CD8+ T-lymphocyte immune responses to human cytomegalovirus pp65 in persons sharing HLA-A*02 and HLA-B*07 alleles.

CD8(+) T-cell responses to three human cytomegalovirus (CMV) pp65 epitopes were studied in panels of healthy seropositive HLA-A*02/HLA-B*07 individuals, and HLA-A*02 donors mismatched for HLA-B*07. The majority of the latter had significant responses to a HLA-A*02-restricted epitope within the CMV pp65 antigen. By contrast, the strongest responses to CMV in the first group were to HLA-B*07-restricted epitopes. Similar immunodominance of HLA-B*07 over HLA-A*02 was found in two immunocompromised HIV-infected HLA-A*02/HLA-B*07 patients, and in the reconstituting immune system of three stem cell transplant recipients. In vitro stimulation of peripheral blood mononuclear cells (PBMC) from two immunocompetent HLA-A*02/HLA-B*07 individuals indicated that cytotoxic T lymphocyte (CTL) precursors specific for both HLA-A*02 and HLA-B*07 restricted epitopes were present and could be expanded by stimulation with the cognate peptides. However, if stimulation was performed by antigen presenting cells infected with recombinant vaccinia expressing full-length native pp65, only HLA-B*07 epitope-specific cells were seen. In one patient the HLA-B*07 dominance was partially broken by using recombinant vaccinia expressing ubiquitinated pp65, suggesting that enhanced protein processing can reveal weaker immune responses. Our results indicate that CMV-specific cellular immune responses restricted by HLA-B*07 dominate those restricted by HLA-A*02 in both immunocompetent and immunocompromised individuals. This may have significant consequences for the design of epitope-specific vaccines.

Characterization of cytotoxic function of CMV-pp65-specific CD8+ T-lymphocytes identified by HLA tetramers in recipients and donors of stem-cell transplants.

BACKGROUND: Human cytomegalovirus (CMV) is a ubiquitous herpesvirus that is an important complication of bone marrow and allogeneic stem-cell transplant (HSCT). CD8 T-lymphocytes have an important role in immunity against CMV, but correlation between antigen-specific subpopulations of these cells and protection are still unclear. METHODS: Flow analysis with fluorescently-conjugated human leukocyte antigen (HLA) class I tetramers (Tet) was used to investigate levels of CMV-specific CD8 T-lymphocytes in peripheral blood monocyte cells (PBMC) samples from HSCT donors and recipients and their ability to produce interferon (IFN)-gamma on stimulation with either CMV antigenic peptide or nonspecific mitogenic stimulation. Chromium release assays were used to evaluate ex vivo CMV-specific cytotoxicity associated with the PBMC samples. RESULTS: Use of Tet in conjunction with fluorescently conjugated anti-T-cell receptor (TCR) beta-chain variable (Vbeta) monoclonal antibodies indicated that the Vbeta repertoires associated with Tet cells seen in two HSCT recipients were similar to the Vbeta repertoires of the Tet cells in their HSCT donors. Significant ex vivo cytotoxicity against peptide-loaded targets was measured from several recipient samples after transplant. However, PBMC from the HSCT donors, even when containing populations of CMV-specific Tet cells capable of secreting IFN-gamma in response to peptide stimulation, possessed no ex vivo CMV-specific cytotoxicity. CONCLUSIONS: We hypothesize that in the setting of the reconstituting immune system of HSCT recipients, CMV reactivation may stimulate a functional change in CMV-specific CD8 T-lymphocytes, rendering them able to directly lyse target cells presenting CMV antigens without in vitro stimulation. These findings have important implications for development of vaccines designed to induce protective cellular immunity to CMV in transplant recipients.