Selective ex vivo expansion of cytomegalovirus-specific CD4+ and CD8+ T lymphocytes using dendritic cells pulsed with a human leucocyte antigen A*0201-restricted peptide.

Adoptive transfer of ex vivo-generated cytomegalovirus (CMV)-specific T lymphocytes may be effective in preventing CMV disease in allogeneic haematopoietic stem cell transplantation (HSCT) recipients. We developed a procedure for expansion of CMV-specific T lymphocytes based on the antigen-presenting function of donor dendritic cells (DCs), pulsed with a human leucocyte antigen A*0201-restricted pp65 nonamer peptide. CMV-specific T lymphocytes were identified following induction of interferon gamma (IFN-gamma) secretion prompted by peptide exposure. Both CD8+ and CD4+ CMV-specific T lymphocytes were selectively produced in these cultures and showed CMV-restricted cytotoxicity. The simultaneous and selective expansion of CD4+ and CD8+ CMV-specific lymphocytes might be instrumental for more efficient in vivo function of infused CMV-specific lymphocytes.

Evaluation of breast tumour cell contamination in the bone marrow and leukapheresis collections by RT-PCR for cytokeratin-19 mRNA.

There is considerable interest in an autologous transplantation (AT) programme for patients with high-risk breast cancer; however, the issue of the incidence of occult bone marrow (BM) micrometastasis at diagnosis, and the cancer contamination of peripheral blood stem cell (PBSC) collections used for haematological rescue, is still debated. The presence of BM micrometastasis was evaluated in bilateral BM biopsies obtained at diagnosis of 33 patients with stage II/IIIA breast cancer using: (i) a 'nested' reverse transcriptase-polymerase chain reaction (RT-PCR) assay for cytokeratin 19 (K19) mRNA, (ii) histology, and (iii) immunohistochemistry (IHC) analysis with a panel of three monoclonal antibodies. The RT-PCR assay only was used to determine contamination of PBSC collections obtained after priming with recombinant human granulocyte-colony stimulating factor (rhG-CSF). K19 transcripts in one or both BM samples were detected in 48% of patients at diagnosis, with an overall 85% concordance with the results of IHC analysis. On the other hand, 56% of PCR- and IHC-positive BM samples were diagnosed as 'normal' on histological analysis. 57% of patients showed K19 mRNA in at least one PBSC collection; the possibility to have contaminated PBSC collections was significantly higher in patients with K19 positivity in BM at diagnosis. In four patients who had shown K19 positivity in BM and in PBSC collections, immunoselected CD34+ cells used for haematological rescue were K19-negative. There was a trend towards longer relapse free survival (RFS) in patients transplanted with K19-negative PBSC collections as compared to the others. In conclusion, a substantial proportion of patients with high-risk non-metastatic breast cancer present occult BM micrometastasis at diagnosis and also show cancer contamination of PBSC collections used for AT. These might represent a category of patients with poorer prognosis after AT, and possible candidates for more intensive and/or alternative therapeutic regimens, including AT with purged PBSCs.