37: Cytotoxic T Lymphocytes (CTL) Specific for Multiple Viruses Can Be Generated From Umbilical Cord Blood for Adoptive Immunotherapy (2008)

Umbilical cord blood (UCB) transplantation is a promising alternative source of hematopoietic stem cells for patients lacking HLA-matched donors. Nearly 60% of UCB transplants to date have been performed on minority individuals for whom an unrelated donor was not available; moreover, the naïve phenotype of UCB cells is responsible for the lower incidence and reduced severity of GvHD in these patients. Still, relatively low cell numbers in UCB grafts have lead to delayed immune reconstitution and higher mortality due to infection. Reactivation of latent viruses such as CMV and EBV are particularly problematic, as is overt infection from adenovirus (Adv). Previous studies have shown that prophylactic adoptive immunotherapy with peripheral blood-derived CTL directed against EBV, CMV and Adv can effectively prevent the clinical manifestations of these viruses after hematopoietic stem cell transplant. We now hypothesize that generating virus-specific CTL from UCB for adoptive immunotherapy will restore anti-viral immunity and reduce viral infection post UCB transplant. Our aim was to generate multi-virus specific CTL from UCB mononuclear cells using a clinical-grade recombinant adenovirus type 5 vector pseudotyped with a type 35 fiber carrying a transgene for CMVpp65. With this Ad5f35pp65 vector we transduced UCB-derived dendritic cells to use as antigen presenting cells to stimulate virus-specific CTL followed by 2 rounds of weekly stimulation with autologous UCB-derived EBV-lymphoblastoid cell lines (LCL) transduced with the same vector. After 3 rounds of stimulation, 5 CTL cultures contained a mean of 87% (range 81–94%) CD8+ve and a mean of 26% (range 12–40%) CD4+ve cells. Evaluable CTL lines showed significant cytotoxicity in chromium release assays against non-transduced autologous LCL and LCL infected with the Ad5f35pp65 and Ad5f35 vectors. The observed cytotoxicity was specific because transduced and non-transduced MHC-mismatched LCL were not killed. ELISPOT assays on CTL demonstrated a mean of 260 (range 45–694) and 47 (range 0–128) numbers of cells secreting IFNγ following incubation with CMVpp65 and Adv hexon peptides respectively. No significant response to CMVIE1 peptides was demonstrated. These results show that, despite the generally naïve nature of UCB lymphocytes, multi-virus-specific responses can be expanded in vitro, and could potentially be used clinically in UCBT patients who develop infectious complications prior to immune reconstitution. Umbilical cord blood (UCB) transplantation is a promising alternative source of hematopoietic stem cells for patients lacking HLA-matched donors. Nearly 60% of UCB transplants to date have been performed on minority individuals for whom an unrelated donor was not available; moreover, the naïve phenotype of UCB cells is responsible for the lower incidence and reduced severity of GvHD in these patients. Still, relatively low cell numbers in UCB grafts have lead to delayed immune reconstitution and higher mortality due to infection. Reactivation of latent viruses such as CMV and EBV are particularly problematic, as is overt infection from adenovirus (Adv). Previous studies have shown that prophylactic adoptive immunotherapy with peripheral blood-derived CTL directed against EBV, CMV and Adv can effectively prevent the clinical manifestations of these viruses after hematopoietic stem cell transplant. We now hypothesize that generating virus-specific CTL from UCB for adoptive immunotherapy will restore anti-viral immunity and reduce viral infection post UCB transplant. Our aim was to generate multi-virus specific CTL from UCB mononuclear cells using a clinical-grade recombinant adenovirus type 5 vector pseudotyped with a type 35 fiber carrying a transgene for CMVpp65. With this Ad5f35pp65 vector we transduced UCB-derived dendritic cells to use as antigen presenting cells to stimulate virus-specific CTL followed by 2 rounds of weekly stimulation with autologous UCB-derived EBV-lymphoblastoid cell lines (LCL) transduced with the same vector. After 3 rounds of stimulation, 5 CTL cultures contained a mean of 87% (range 81–94%) CD8+ve and a mean of 26% (range 12–40%) CD4+ve cells. Evaluable CTL lines showed significant cytotoxicity in chromium release assays against non-transduced autologous LCL and LCL infected with the Ad5f35pp65 and Ad5f35 vectors. The observed cytotoxicity was specific because transduced and non-transduced MHC-mismatched LCL were not killed. ELISPOT assays on CTL demonstrated a mean of 260 (range 45–694) and 47 (range 0–128) numbers of cells secreting IFNγ following incubation with CMVpp65 and Adv hexon peptides respectively. No significant response to CMVIE1 peptides was demonstrated. These results show that, despite the generally naïve nature of UCB lymphocytes, multi-virus-specific responses can be expanded in vitro, and could potentially be used clinically in UCBT patients who develop infectious complications prior to immune reconstitution.