Adoptive T Cell Therapy for Progressive Multifocal Leukoencephalopathy Using Sequential Ex-Vivo Stimulation with JCV Peptide Pulsed Dendritic Cells and Anti-CD3/CD28

Progressive multifocal leukoencephalopathy (PML) is a rare but deadly demyelinating disease of the central nervous system caused by reactivation of the polyomavirus JCV. It develops in the setting of severe cellular immune deficiency such AIDS, hematologic malignancies, recipients of hematopoietic stem cell transplants, and following administration of immunodepleting monoclonal antibodies such as rituximab. Patients who demonstrate an antiviral response characterized by the presence of JCV-specific CD8⁺ cytotoxic T lymphocytes (CTL) exhibit improved outcomes. However, efforts to elicit the expansion of JCV specific T cells using dendritic cells (DCs) pulsed with JCV peptides have been frustrated by the lack of effector cell responses in this setting. We have demonstrated that sequential stimulation with DC based vaccines and ligation of the costimulatory complex using anti-CD3/CD28 results in the dramatic expansion of activated T cells. In this study we sought to examine whether ex vivo stimulation with anti-CD3/CD28 facilitated JCV responses that might be used as adoptive immunotherapy. Methods: Dendritic cells (DCs) were generated from adherent mononuclear cells isolated from normal donors and cultured with rhIL-4 and GM-CSF for 5 days and then matured with TNF for 2 days. The non-adherent cells were cultured with IL-2 as a source of autologous T cells. DCs were pulsed with the VP1p36 and VP1p100 peptides and co-cultured with autologous T cells in the presence of low-dose IL-2. After 10 days, an aliquot of the stimulated T cells were exposed to antiCD3/CD28 antibody coated plates for 2 days. The expression of cell surface molecules, intracellular cytokines and VP1p36/p100 tetramers were assessed by flow cytometry. Results: Sequential stimulation with DCs pulsed with JCV derived peptides and anti-CD3/CD28 resulted in a 3 fold expansion of T cells specific for the JCV derived peptides as compared to that observed with peptide pulsed DCs alone. The addition of anti-CD3/CD28 also resulted in polarization of the T cell population towards a Th1 phenotype as evidenced by an increase in the expression of IFNγ by CD4+(4.1 to 8%, p=0.04) and CD8+ cells (2.72 to 7.7%,p=0.035) T cells. In contrast, expression of IL-10 on CD4+ and CD8+ T cells decreased following anti-CD3/CD28 stimulation. Consistent with these findings, sequential stimulation with DCs pulsed with peptides and anti-CD3/CD28 resulted in an increase in CD4/CD25/CD69+ cells (5 to 14.9%; p=0.01). Conversely, sequential stimulation with the DC-JCV peptide vaccine and anti-CD3/CD28 did not result in an increase in regulatory T cells as represented by CD4/CD25/FoxP3+ cells (7.0% vs. 5.9%, respectively (p>0.5)). Discussion: These findings suggest that sequential stimulation with a DC-peptide based vaccine for PML and anti-CD3/CD28 ligation results in a significant increase in JCV specific T cells that exhibit an activated phenotype. The stimulated T cell population is characterized by the expression of Th1 cytokines, activation markers, and a lack of concurrent regulatory T cell expansion. This approach provides a potential strategy for the development of adoptive immunotherapy for PML.

No relevant conflicts of interest to declare.