ALBERT

Description: Abstract 2437 Poster Board II-414 Introduction: BK virus (BKV) is a polyomavirus that is ubiquitous in humans, infecting over 85% of normal individuals. After initial infection it persists in a latent state in the urothelium from whence it can reactivate causing disease in immunocompromised patients. BKV is a cause of haemorrhagic cystitis after allogeneic SCT and is emerging as one of the major causes of graft loss after renal transplant. Current treatment is limited to reduction of immunosuppression as possible. Aim: To develop a method for production of a T cell product with BKV specificity from normal donors for use in adoptive immunotherapy post hemopoietic stem cell transplantation. Methods: Peripheral blood mononuclear cells (PBMC) or monocyte derived dendritic cells (mo-DC) were pulsed with mixes of overlapping peptides covering the 5 BKV proteins (VP1, VP2, VP2 isoform 3, large T antigen (LTA) and small T antigen (sTA)). Mo-DC were produced by isolating monocytes by adherence to plastic and culturing for 7 days in GM-CSF and IL-4 containing media. On day 6 mo-DC were matured by the addition of TNF. T cells were stimulated on day 1 and 7 with peptide pulsed PBMC or mo-DC and cultured for 21 days with increasing doses of IL-2 from day 7. The cellular product was then analysed for phenotype, BKV specificity and functionality by examining cytokine production and cytotoxicity. Results: Cellular proliferation was seen in all of 10 normal donors with a mean increase of 6.4 fold in total cell number. All cellular products were 〉84% CD3 positive (Mean 96%, SEM 1.3) with CD4 and CD8 ratio varying significantly between individual donors (CD4 range 9.7 to 97.5%, mean 70.79; CD8 range 0.8 to 77.0%, mean 23.3). Cells were of memory phenotype, being CD28+ (mean 86.1%, SEM 6.5), CD45RO+ (mean 84.1%, SEM 5.9) and a variable proportion were of central memory phenotype (CD62L+ mean 21.6%, range 6.9 to 55.0). Cytokine responses to stimulation with BKV peptides could be elicited in 5 of 6 evaluable donors. Multiple cytokines were produced by the responding cells: IFN-γ (mean 29.9% of CD3 cells, range 4.5 to 78.8), TNF (mean 19.9%, range 2.7 to 63) and IL-2 (mean 12.8%, range 1.2 to 37.8). Cytokine responses were seen in both CD4 and CD8 cells and showed significant individual variation. VP1 and LTA specific cells dominated most cultures while a smaller percentage of the cells were specific for VP2, VP2 isoform 3 and sTA. CD8 specificity was mainly confined to a single protein whereas CD4 responses tended to be of lower magnitude but broader specificity. Cultures exhibited cytotoxic activity with the lysis of BKV antigen coated target cells in a pattern that correlated with the presence of CD8 positive cytokine producing cells (up to 78.9% specific lysis at effector to target ratio of 20:1). Discussion: The clinical utility of this product remains to be determined. Potential uses include prophylaxis and therapy of reactivation of BK virus after hemopoietic stem cell or renal transplantation. This method for large-scale expansion of BKV specific CTL could be utilised for analysis of BKV targeted immune responses and epitope identification. Disclosures: No relevant conflicts of interest to declare.