Abstract

Cytomegalovirus (CMV) infections remain a major cause of morbidity and mortality following allogeneic hematopoietic cell transplantation. For recipients of HLA haplotype disparate grafts, the risk of CMV infection is especially high and extends late into the post-transplant period. Early clinical trials indicate that adoptive transfer of ex vivo generated CMV-specific T-cells from the donor can be effective in the treatment and prevention of such infections. However, because seropositive donor T-cells sensitized with autologous infected or antigen loaded APCs regularly exhibit a repertoire of CMV-specific T-cells restricted by one or two immunodominant HLA alleles, such T-cells will only be effective if these immunodominant alleles are shared by the infected host. For example, among donors inheriting HLA-B*0702, CD8+ T-cell responses to CMV-pp65 peptides are usually, and often exclusively, restricted by this allele.

To address this limitation, we tested whether artificial antigen-presenting cells, consisting of murine 3T3 cells transduced to express human B7.1, LFA1, ICAM1, β 2M and HLA-A*0201 alpha chain (Papanicolaou et al., Blood 2003), could be used to generate CMV-specific T-cells restricted by this allele in a series of normal donors inheriting HLA-A*A0201 and HLA-B*0702. Accordingly, T-cells were sensitized in vitro with either autologous monocyte-derived DCs or AAPCs expressing HLA-A*A0201, each loaded with a pool of 138 overlapping pentadecapeptides spanning the CMV-pp65 protein. Thereafter, specificity of responding T-cells was identified by mapping epitopes using an intersecting matrix of peptide subpools and measuring T-cells producing IFN-γ following secondary restimulation. HLA restriction was identified by analysis of T-cell cytotoxic responses against panels of EBV BLCL sharing single HLA alleles with the donor and loaded with the targeted peptide. Findings were confirmed by quantitation of T-cell binding tetramers containing targeted epitopes bound to either HLA-A*0201 or B*0702. In each of the donors, sensitization with DCs loaded with the peptide pool resulted in the generation of CMV-pp65-specific T-cells specific for epitopes predominantly or exclusively presented by HLA-B*0702. In contrast, sensitization with peptide pool loaded AAPC led to the generation of high numbers of T-cells recognizing separate CMV-pp65 epitopes in the context of HLA-A*A0201. These HLA-A*0201-restricted T-cells were also able to lyse peptide-loaded BLCLs or PBMCs expressing HLA-A*0201. Taken together, these studies demonstrate that AAPCs expressing single HLA alleles can be used to generate fully functional T-cells specific for epitopes presented by subdominant HLA alleles. This strategy may thus permit generation of virus-specific T-cells of desired HLA-restriction for adoptive immunotherapy in HLA-disparate transplant recipients.

Author notes

Corresponding author