Optimizing Culture Conditions for Ex Vivo Expansion of Virus Specific T Cells.

Adoptive immunotherapy with virus specific CD8+ cytotoxic T lymphocytes (CTL) is currently an option for treatment of viral infections after allogeneic stem cell transplantation. A major obstacle for clinical grade production of virus specific CTL is to retain the antigen specific clones, which can be easily deleted during ex vivo expansion. Manipulation of CD3/CD28 engagement, depletion of T regulatory (Treg) cells before expansion and prestimulation with antigen presenting cells were explored in this study. Mononuclear cells were obtained from HLA-A2+ donors by leucapheresis. Lymphocytes were enriched by elutriation, stimulated with anti-CD3/anti-CD28^high Dynabeads or Dynabeads® ClinExVivo™ CD3/CD28 and cultured for 10 days in CellGro media supplemented with human AB serum and IL-2. Virus specific CD8+ CTL were quantified by flow using pentamer staining. By using anti-CD3/ anti-CD28^high Dynabeads a more balanced expansion of CD4+ and CD8+ subsets was obtained, while Dynabeads ClinExVivo CD3/CD28 allowed a higher expansion of CD8+ subset and therefore a higher expansion of the virus specific CTL. A high Dynabead: T cell ratio (3:1) deleted completely the virus specific CTL. By reducing this ratio we could retain the virus specific CTL after expansion. No benefits were observed by adding extra Dynabeads during expansion. It is known that Treg cells can inhibit expansion of all T cell subsets. By depleting Treg cells with CD25 Dynabeads prior to T cell expansion, we observed a significantly increased expansion of all T cells, including virus specific CTL. In some patients low numbers of virus specific CTL can be detected. To increase the numbers of antigen specific CTL we have included a prestimulation step using peptide-loaded mononuclear cells prior to expansion with Dynabeads, which gave more than 3000- fold expansion of virus specific CTL. We are currently exploring the phenotype profile of expanded CTL (CD62L, CCR7, CD57, CD27, CD28 and CTLA-4), functionality (cytokine secretion and proliferative capacity) and cytotoxicity. This protocol can be upgraded to clinical grade production of virus specific CTL for treatment of viral infections in immunocompromised patients.