Abstract
Mantle cell lymphoma (MCL) is an aggressive non-Hodgkin’s lymphoma with the worst long-term prognosis of any NHL subtype. Current therapeutic options are unsatisfactory. MCL patients’ malignant B cells are ineffective antigen-presenting cells (APCs), perhaps resulting from low level expression of the immune co-stimulatory molecules that are essential to activate T cells upon interaction with the T-cell receptor. The MCL cells can be engineered to be effective APCs and thereby function as a therapeutic cellular vaccine in combination with chemotherapy and/or stem cell transplantation to eradicate residual disease. However, primary MCL cells are difficult targets for gene transfer by both viral and non-viral methodologies. Ligation of CD40 resulting from co-culturing with hCD40L expressing murine fibroblasts was shown to be superior to a panel of other immune stimulants and cytokines in upregulating co-stimulatory markers and inducing anti-tumor T cell responses (Hoogendoorn et al. 2005). We now report on a technology platform, based on electroporation of mRNA for CD40L, for the introduction of CD40L protein expression and subsequent induction of immune co-stimulatory molecules by MCL tumor cells. Primary MCL malignant B cells were obtained from patients’ lymph node biopsies by mechanical dissociation, placed in single cell suspension and cryopreserved prior to modification. Full-length 5′-end capped hCD40L mRNA transcript was generated by in vitro transcription with a commercially available T7 polymerase kit. The transfected MCL cells were immunostained with fluorophore-conjugated monoclonal antibodies against hCD40L, hCD80 and 86 then analyzed by FACS. Data showed hCD40L could be detected in ≥ 80% of the transfected MCL cells as early as 2 hrs post transfection. At 3 days post manipulation, hDC40L expression could be detected on approximately 30% of the transfected MCL cells. Cell viability remained at approximately 80% during the 3 day in vitro culturing. FACS analysis of the immune co-stimulatory molecules revealed that forced expression of hCD40L caused an up-regulation of CD80/86, which was increased approximately 10 fold compared to the expression levels in naïve, non modified cells. The increased expression level of CD80/86 was maintained for 3 days. Furthermore, when the hCD40L modified MCL cells were mixed with allogeneic PBMC, they stimulated IFN-γ production at a level 4 fold higher than was observed with naïve, non modified MCL cells mixed with allogeneic PBMC. This provides proof-of-concept that MCL cells modified by mRNA-hCD40L transfection have the potential to be used as a cellular vaccine. Such transduced cells function to protect animals from tumor challenge. The process can be scaled up to produce >2×1010 modified tumor cells. This simple, non-viral cell manipulation system is practical and will be a useful tool for immunotherapy of human hematopoietic malignancies such as MCL.
Immune evasion of mantle cell lymphoma: expression of B7-H1 leads to inhibited T-cell response to and killing of tumor cells