Functional comparison of T cells recognizing cytomegalovirus pp65 and intermediate-early antigen polypeptides in hematopoietic stem-cell transplant and solid organ transplant recipients.

The functional status of cytotoxic T lymphocyte (CTL) populations recognizing cytomegalovirus intermediate-early antigen (IE1) and pp65 polypeptides was investigated in peripheral blood mononuclear cells from hematopoietic stem-cell transplant (HSCT) and solid organ transplant recipients. Combined flow-based CD107a/b degranulation/mobilization and intracellular cytokine (ICC) assays using peptide libraries as antigens indicated that a significantly higher proportion of pp65-specific CTLs were in a more mature functional state, compared with IE1-specific CTLs. Degranulation/multiple cytokine ICC assays also indicated that a significantly higher proportion of pp65-specific than IE1-specific CTLs secreted both interferon- gamma and tumor necrosis factor- alpha and possessed greater cytotoxic potential. These results support our earlier findings of functional differences between CTLs recognizing individual epitopes within the IE1 and pp65 antigens in healthy donors and HSCT recipients and extend them to a broader array of human leukocyte antigen-restricted responses to those antigens. We also provide evidence of a relationship between cytotoxic function and the ability of cytomegalovirus-specific CTLs to secrete multiple cytokines.

Cross-reactive CTL recognizing two HLA-A*02-restricted epitopes within the BK virus and JC virus VP1 polypeptides are frequent in immunocompetent individuals.

Two HLA-A*02-restricted epitopes have been identified within the VP1 polypeptide of a human polyomavirus, BK virus, which is associated with polyomavirus-associated nephropathy in kidney transplant patients. Immunization of transgenic mice with recombinant modified vaccinia Ankara expressing BKV VP1 (rMVA-BKV VP1) elicited functional CTL populations recognizing the sequences LLMWEAVTV (amino acids residues 108-116, BKV VP1p108) and AITEVECFL (residues 44-52, BKV VP1p44) and cross-reactive to the previously described JC virus VP1 homologs. Flow-based analyses of PBMC from a panel of thirty healthy HLA-A*02 human volunteers indicated that the majority of these subjects harbored functional CTL populations recognizing the BKV epitopes and cross-reactive with the JCV homologs. CTL recognizing the JCV VP1p100 and JCV VP1p36 epitopes have previously been associated with prolonged survival in progressive multifocal leukoencephalopathy patients. These findings suggest that infection with BKV or JCV could potentially induce cross-protective T-cell immunity against diseases associated with these viruses.

Cross-reactivity of T lymphocytes recognizing a human cytotoxic T-lymphocyte epitope within BK and JC virus VP1 polypeptides.

A transgenic mouse model was used to identify an HLA-A*02-restricted epitope within the VP1 polypeptide of a human polyomavirus, BK virus (BKV), which is associated with polyomavirus-associated nephropathy in kidney transplant patients. Peptide stimulation of splenocytes from mice immunized with recombinant modified vaccinia virus Ankara expressing BKV VP1 resulted in expansion of cytotoxic T lymphocytes (CTLs) recognizing the sequence LLMWEAVTV corresponding to amino acid residues 108 to 116 (BKV VP1p108). These effector T-cell populations represented functional CTLs as assessed by cytotoxicity and cytokine production and were cross-reactive against antigen-presenting cells pulsed with a peptide corresponding to the previously described JC virus (JCV) VP1 homolog sequence ILMWEAVTL (JCV VP1p100) (I. J. Koralnik et al., J. Immunol. 168:499-504, 2002). A panel of 10 healthy HLA-A*02 human volunteers and two kidney transplant recipients were screened for T-cell immunity to this BK virus VP1 epitope by in vitro stimulation of their peripheral blood mononuclear cells (PBMC) with the BKV VP1p108 peptide, followed by tetramer labeling combined with simultaneous assays to detect intracellular cytokine production and degranulation. PBMC from 4/10 subjects harbored CTL populations that recognized both the BKV VP1p108 and the JCV VP1p100 peptides with comparable efficiencies as measured by tetramer binding, gamma interferon production, and degranulation. CTL responses to the JCV VP1p100 epitope have been associated with prolonged survival in progressive multifocal leukoencephalopathy patients (R. A. Du Pasquier et al., Brain 127:1970-1978, 2004; I. J. Koralnik et al., J. Immunol. 168:499-504, 2002). Given that both human polyomaviruses are resident in a high proportion of healthy individuals and that coinfection occurs (W. A. Knowles et al., J. Med. Virol. 71:115-123, 2003), our findings suggest a reinterpretation of this protective T-cell immunity, suggesting that the same VP1 epitope is recognized in HLA-A*02 persons in response to either BK or JC virus 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.