Allogeneic Cytokine-Induced Killer Cells Traffick to GVHD Target Tissue but Cause Less GVHD, While Retaining Tumor Cytotoxicity and Facilitating Bone Marrow Engraftment.

Abstract

Cytokine-induced-killer cells (CIK), generated from splenocytes in mice and PBMC in human by the timed addition of IFN-γ, anti-CD3 MAbs and IL-2, express both T cell and NK cell markers and have cytotoxic activity against autologous and allogeneic tumors. We previously showed that adoptive transfer of allogeneic CIK in a murine model did not cause severe acute GVHD, leading to full survival. However the precise mechanism of reduced GVHD and retained GVT activity is not fully understood. To this end, we first evaluated the trafficking patterns of luciferase-transgenic CIK in a major MHC mismatched BMT model. In vivo bioluminescence imaging (BLI) showed that both CIK and fresh splenocytes homed to and proliferated initially in secondary lymphoid organs including the spleen, followed by infiltration of the gut and skin by day 7. All mice receiving fresh splenocytes died by day 10. Signal from gut mucosa in animals receiving allogeneic CIK cells was transient, however signals from other sites such as mesenteric lymph nodes were high and persistent. By day 21 allogeneic CIK reinfiltrated intestinal tissue without causing severe GVHD. To further investigate potential changes in the expression of surface molecules in vivo, we transplanted GFP positive fresh splenocytes and GFP positive CIK cells into major mismatched irradiated hosts, and then on day 7 analyzed activation markers, homing molecules, apoptosis related genes, and peripheral tolerance induction molecules such as CTLA-4 and PD-1 by FACS. No significant differences were noted between CIK and splenocytes. However we found that the cell division rate of CIK cells in vivo was much slower than those of splenocytes utilizing a CFSE based cell proliferation assay. This might lead to less GVHD in mice receiving CIK cells. Next we evaluated whether CIK retained tumor killing activity in vivo using A20 luciferase lymphoma cells into lethally irradiated MHC major mismatched receipients subcutaneously. We visualized the reduction of tumor by BLI. GFP positive CIK, which were extracted on day 7 following their transplantation into MHC-mismatched allogeneic recipients, retained tumor cytotoxicity in vitro. Finally, to study whether CIK facilitate BM engraftment, we quantified the absolute number of cells from the peripheral blood in MHC major-mismatched nonmyeloablative BMT. On day 30 mice receiving CIK showed rapid BM engraftment, especially in CD4 (p=0.0007) and CD8 (p<0.0001) subpopulations. In conclusion, allogeneic CIK homed to and expanded in GVHD target organs without causing acute GVHD, retained tumor killing activity and promoted BM engraftment.