Reactivation of endogenous viruses, e. g. CMV, is a major cause of morbidity and mortality after allogeneic hemopoietic stem cell transplantation (allo-HSCT). Risk factors for the reactivation of CMV include the conditioning and immunosuppressive regimen, T-cell depletion of the graft, the development, and the treatment of acute or chronic graft versus host disease (GvHD). T-cells play an important role in the control of viral reactivation, lack of T-cells often leads to recurrent viral infections. The cytokine secretion assay provides the possibility to screen for CMV-reactive cells in a simple FACS assay by detection of interferon-gamma (IFN-gamma) secreting T-cells after stimulation of the cells with CMV-antigen. In addition IFN-g secreting cells can be enriched using the same assy. We have recently optimized enrichment to achieve 80-95% purity of IFN-gamma secreting T-cells.

In order to further optimize stimulation, several CMV-antigens were tested: While viral lysate provides a broad activation of CMV-specific T-cells, recombinant proteins like pp65 may offer more reproducible conditions. Comparison of viral lysate to pp65 regarding activation and enrichment of CMV-specific T-cells showed a possible HLA-dependency of pp65-performance: in HLA-A2-positve donor’s stimulation by pp65 was equal to stimulation with lysate, while in other donors pp65 was less effective. Interestingly, stimulation with lysate yielded mainly CD4-positive T-cells, whereas stimulation with pp65 mainly yielded CD8-positive T-cells.

Five patients, who reactivated CMV after allo-HSCT, were monitored closely for CMV-reactive cells to date. Initially all patients had no or reduced numbers of CMV-reactive T-cells. After one year, in 4 patients CMV-responsiveness was restored, none of these reactivated CMV more than twice. One patient had recurrent CMV-reactivations after HSCT, starting as early as day +20 after HSCT despite of GCV-treatment. Thus, CMV-specific enriched donor T-cells were transfused to control the reactivation. In an effort to minimize the risk of GvHD and to maximize protection from CMV-disease after allo-HSCT, we applied immunomagnetic selection for the isolation of CMV-reactive T-cells from the sero-positive donor. The persistence and proliferation of the transfused CMV-specific T-cells was monitored. During the first 4 weeks the percentage of CMV-reactive T-cells raised from undetectable levels to 3.5%, while CMV-antigenemia declined. After 3 weeks a complete clearance of CMV was achieved. No GvHD occurred, despite a mismatch in HLA-DR between donor and recipient. These preliminary data suggest the feasibility and safety of the transfusion of CMV-specific T-cells after immunomagnetic selection for the treatment of CMV-reactivation.

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