RNA-loaded CD40-activated B cells stimulate antigen-specific T-cell responses in dogs with spontaneous lymphoma

Abstract

Cell-based vaccination strategies to induce functional tumorspecific T cells in cancer patients have focused on using autologous dendritic cells. An alternative approach is to use RNA-loaded CD40 activated B cells (CD40-B) that are highly efficient antigen-presenting cells capable of priming naive T cells, boosting memory T-cell responses and breaking tolerance to tumor antigens. The use of tumor RNA as the antigenic payload allows for gene transfer without viruses or vectors and permits major histocompatibility complex (MHC)-independent, multiple-antigen targeting. Here, we use CD40L transfected K562 cells to generate functional CD40-B cells from the peripheral blood of humans and dogs. Testing of RNA-loaded CD40-B cells in dogs allows not only for its development in veterinary medicine but also for determination of its safety and efficacy in a large animal model of spontaneous cancer prior to initiation of human clinical trials. We found that CD40-B cells from healthy humans, healthy dogs and tumor-bearing dogs express increased levels of immune molecules such as MHC and CCR7. Moreover, RNAloaded CD40-B cells induce functional, antigen-specific T cells from healthy dogs and dogs with lymphoma. These findings pave the way for immunotherapy trials using tumor RNA-loaded CD40-B cells to stimulate antitumor immunity in a large animal model of spontaneous neoplasia. Gene Therapy (2008) 15, 955-965;

Evidence of an oncogenic gammaherpesvirus in domestic dogs

“…Cryopreserved, single cell suspensions of malignant lymph nodes from 2 dogs with positive ISH-EBER results and 1 dog with a low p18 antibody titer were thawed and cultured with lethally irradiated KtCD40L cells as previously described for the expansion of healthy canine B cells from PBMCs (Mason et al, 2008). Efforts to expand cryopreserved, malignant canine B cells in the absence of CD40L were unsuccessful.…”

“…EBV naturally infects only humans and previous efforts to understand its role in lymphomagenesis have relied upon the use of species-specific LCV of non-human primates to approximate EBV infection and pathogenesis. The domestic dog develops spontaneous lymphoma that shares biological, behavioral, genetic and cytogenetic similarities to DLBCL and Burkitt-like lymphoma subtypes in humans and is widely recognized as a model to evaluate the safety and efficacy of novel therapies for these disease entities in humans (Bergman et al, 2003; Breen and Modiano, 2008; Fournel-Fleury et al, 1997; Lin et al, 2008; MacEwen, 1990; Mason et al, 2008; Paoloni and Khanna, 2008; Paoloni et al, 2009; Thomas et al, 2003). Here we use ELISA, ELISA blocking experiments, Western blot and IFA to show that dogs develop specific serological responses against viral capsid antigens of an EBV-like gammaherpesvirus and thus extend previous findings of EBV-like seropositivity in dogs (Chiou et al, 2005; Milman, Smith, and Erles, 2011).…”

“…Cryopreserved malignant lymphocytes were thawed and cultured with lethally irradiated K562 cells transfected with human CD40L (KtCD40L) at a ratio of 5:1 in the presence of recombinant canine IL-4 (R&D Systems, Minneapolis, MN) and cyclosporine as previously described (Mason et al, 2008). Cells were re-stimulated every 5-7 days with fresh, lethally irradiated KtCD40L.…”

The properties of human CD40-activated B cells as antigen-presenting cells are not affected by PGE2

“…CD40-activated B cells are currently being studied as an alternative type of APC for cellular vaccines (23)(24)(25)(26). Most importantly, they can be easily expanded ex vivo from peripheral blood of cancer patients (27).…”

Inhibition of Protein Geranylgeranylation Specifically Interferes with CD40-Dependent B Cell Activation, Resulting in a Reduced Capacity To Induce T Cell Immunity

“…We and others have previously shown that B cells acquire potent Ag-presenting function following crosslinking of CD40 (5,6). Activation via CD40 is probably the strongest stimulus for the induction of B cell-mediated Ag presentation and cytokine production (7).…”