Depletion of alloreactive T cells via CD69: implications on antiviral, antileukemic and immunoregulatory T lymphocytes.

Selective depletion of alloreactive T cells from stem-cell allografts should abrogate graft-versus-host disease while preserving beneficial T cell specificities to facilitate engraftment and immune reconstitution. We therefore explored a refined immunomagnetic separation strategy to effectively deplete alloreactive donor lymphocytes expressing the activation antigen CD69 upon stimulation, and examined the retainment of antiviral, antileukemic, and immunoregulatory T cells. In addition to the CD69high T cell fraction, our studies retrieved two T cell subsets based on residual CD69 expression. Whereas, truly CD69(neg) cells were devoid of detectable alloresponses to original stimulators, CD69-low (CD69low)-expressing T cells elicited significant residual alloreactivity upon restimulation. In interferon-gamma enzyme linked immunospot assays, anti-cytomegalovirus and anti-Epstein-Barr virus responses were preserved at significant numbers among CD69neg T lymphocytes. Accordingly, T cells recognizing the leukemia-associated Wilm's tumor-1 antigen were still detectable in the CD69neg subset. However, antiviral and antileukemic specificities were also consistently found within CD69low T cells, suggesting that memory-type donor T cells were partially captured due to residual CD69 expression. Finally, CD4+CD25+ Foxp3+ immunoregulatory T cells did not upregulate CD69 upon allogeneic stimulation. Our data suggest that CD69-mediated removal of alloreactivity can result in efficient allodepletion, but may partially affect the persistence of antiviral and antileukemic donor memory specificities captured among CD69low-expressing lymphocytes.

Microcell-mediated transfer of chromosome 4 into HeLa cells suppresses telomerase activity.

Telomerase activity can be detected in most human cancers and immortal cell lines. In contrast, the lack of telomerase activity in normal diploid fibroblasts has been correlated with progressive reduction of telomere lengths to critically short sizes followed by the cessation of cell division and the onset of senescence. Several investigators have provided evidence for the localization of a telomerase suppressor gene on chromosome 3. The aim of our study was to determine whether other chromosomes are involved in telomerase repression. Beside human chromosome 3 (serving as positive control), chromosomes 4, 6, and 11 were introduced into HeLa cells via microcell-mediated chromosome transfer. Telomerase activity from different hybrid cell lysates was determined at an early time point after fusion using a Telomerase ELISA kit. Strong repression of telomerase activity was only found in a subset of HeLa hybrids in which chromosome 3 or chromosome 4 had been introduced. Telomerase suppression induced by chromosome 3 or 4 transfer was paralleled by a high frequency (30% or 43%, respectively) of a senescent-like phenotype. Chromosomes 6 and 11, the functional loss of which is also implicated in cervical cancer, had no effect. These results indicate that normal human chromosomes 3 and 4 carry a gene or genes that suppress telomerase activity and induce cellular senescence in HeLa cells.

HPV16 L1E7 chimeric virus-like particles induce specific HLA-restricted T cells in humans after in vitro vaccination.

Cervical cancer has been shown to be highly associated with human papillomavirus (HPV) infection. The viral oncogenes E6 and E7 are constantly expressed by the tumor cells and are therefore targets for immunotherapy. In the present study we investigated the potential of HPV16 L1E7 chimeric virus-like particles (CVLP) to activate specific cytotoxic T lymphocytes in human blood donors. CVLP were expressed by recombinant baculovirus and purified. Direct incubation of freshly isolated peripheral blood lymphocytes (PBL) with CVLP resulted in induction of proliferation and growth of T cell lines. To enhance antigen presentation we also loaded dendritic cells with CVLP and used them to activate naive T cells. Growing cell lines were mainly CD3 positive (>95%) with a predominant CD4-positive and a minor CD8-positive component. Analysis of Tcell specificity was carried out by an interferon-gamma ELISpot assay. Dendritic cells pseudoinfected with CVLP or pulsed with human leukocyte antigen (HLA)-A*0201-restricted peptide E7(11-20) or with a newly identified HPV16 peptide L1(323-331) were used as stimulator cells. T cells responsive to CVLP were found in the cultures with frequencies of 0.5%-0.7%. Frequencies to peptides were around 0.1%. These T cells had cytolytic activity toward autologous B-lymphoblastic cell lines either pseudoinfected with CVLP or pulsed with HLA-A*0201-restricted peptides. They also lysed the HPV16- and HLA-A*0201-positive cervical cancer cell line CaSki, whereas HLA-A*0201-negative SiHa cells were not lysed. We conclude from our data that CVLP show promise for a therapeutic vaccine in patients with HPV16-positive cervical intraepithelial neoplasia lesions or cervical cancer.