Abstract
Abstract 2430
B-cell type chronic lymphocytic leukemia (CLL), an incurable disease of unknown etiology, results from the clonal expansion of a CD5+CD19+ B lymphocyte. Our previous study indicated a key role of autologous T cell activation in the successful engraftment and proliferation of CLL cells in NOD/SCID/gcnull (NSG) mice. This T-cell activation, however, was eventually detrimental as it led to CTL generation and elimination of the leukemic clone. By analyzing a panel of human cytokines in the sera of mice treated in different ways in the xenogeneic model, we identified sCD40L as a molecule that correlated with the level of CLL proliferation regardless of T-cell abundance.
To determine if sCD40L was really responsible for CLL proliferation in vivo, we preconditioned NSG mice with human sCD40L by delivering into mouse liver an expression vector coding for hCD40L using the hydrodynamic gene delivery technique; this permitted hepatocytes to produce and secrete the soluble protein. This technique uses hydrodynamic force, generated by an injection of a large volume (10% of body weight) of DNA solution (30μg/ml) over 5–7 seconds through a blood vessel (tail vein), to permeabilize the capillary endothelium of the liver and generate “pores” in the plasma membrane of the surrounding parenchymal cells, thereby allowing DNA to reach the cell interior. With time, membrane pores close, trapping these molecules inside. At that point, transfected cells transiently produce the molecule encoded by the vector.
We generated a CD40L construct consisting of the soluble part of CD40L and the trimerization domain of adiponectin (ADPN-CD40L). The adiponectin domain creates a trimer which can also dimerize to a hexamer, creating a multimeric form with higher biological activity. Furthermore, glycosylation of adiponectin increases the stability of the molecule.
After hydrodynamic gene delivery of the ADPN-CD40L expression vector, high levels of ADPN-CD40L (> 5ng/ml) were found in the sera of treated mice for over two months. Next, CFSE-labeled CLL PBMCs were injected in these mice. Untreated mice and mice co-injected with allogeneic monocytes served as negative and positive controls. Ten days after CLL cell injection into mice with circulating ADPN-CD40L, we documented leukemia cell proliferation in the blood and spleen by CFSE dilution for every CLL sample studied, although the degree of proliferation varied among samples. Nevertheless, proliferation could be extensive with leukemic cells from some cases undergoing more than 6 rounds of division. In contrast, there was no proliferation in untreated mice or negligible/minimal CLL cell proliferation in mice receiving monocytes, probably because the time point was too early to permit autologous T-cell expansion and consequentially CLL cell proliferation. Currently the effects of other cytokines, alone or in combination with ADPN-CD40L, on engraftment and long term growth of CLL cells in vivo are being studied.
Thus, this approach appears to reduce/eliminate a down side of our initial model whereby necessary but uncontrolled T-cell expansion to alloantigens generated GVHD and GVL making the model short term. This new approach may lead to a long term model that would allow more complex preclinical studies or would be a better tool to study the basic biology of CLL cells in vivo.
Disclosures:
No relevant conflicts of interest to declare.
Mechanisms of tolerance induction: blockade of co–stimulation