Pig to rat cell transplantation: reduced cellular and antibody responses to xenografts overexpressing PD-L1.

BACKGROUND: Programmed death-1 (PD-1) costimulation acts as a negative regulator of T-cell responses to allografts. However, the role of the PD-1 pathway in xenotransplantation is not well defined yet. We have shown previously that human in vitro T-cell responses to porcine transfectants overexpressing PD-Ligand1 (L23-PD-L1 cells) are remarkably weak. In this report, we asked whether the PD-1/PD-L1 pathway has the potential to diminish xenogeneic immune responses also in vivo. METHODS: L23-PD-L1 or mock transfected control cells (L23-GFP) were transplanted under the kidney capsule of rats. The occurrence of kidney-infiltrating rat leukocytes and the induction of anti-pig antibodies were monitored in grafted animals. RESULTS: Assessment of cellular infiltrates revealed similar numbers of macrophages in kidneys grafted with L23-PD-L1 or L23-GFP control cells. However, the level of MHC class-II molecules was reduced on macrophages responding to L23-PD-L1 grafts, suggesting a lower state of activation. Furthermore, less T cells were found in kidneys receiving L23-PD-L1 cells. In addition, the titers of induced anti-pig antibodies were significantly lower in rats grafted with L23-PD-L1 cells. CONCLUSIONS: These data suggest that signals triggered by PD-1-PD-L1 interaction interfere with activation pathways involved in the induction of cellular and antibody-mediated immune responses to xenografts in vivo. Targeting of PD-1 and/or PD-L1 may be a promising approach for immune modulation after xenotransplantation.

Exchange of cytosolic content between T cells and tumor cells activates CD4 T cells and impedes cancer growth.

BACKGROUND: T cells are known to participate in the response to tumor cells and react with cytotoxicity and cytokine release. At the same time tumors established versatile mechanisms for silencing the immune responses. The interplay is far from being completely understood. In this study we show contacts between tumor cells and lymphocytes revealing novel characteristics in the interaction of T cells and cancer cells in a way not previously described. METHODS/ FINDINGS: Experiments are based on the usage of a hydrophilic fluorescent dye that occurs free in the cytosol and thus transfer of fluorescent cytosol from one cell to the other can be observed using flow cytometry. Tumor cells from cell lines of different origin or primary hepatocellular carcinoma (HCC) cells were incubated with lymphocytes from human and mice. This exposure provoked a contact dependent uptake of tumor derived cytosol by lymphocytes--even in CD4⁺ T cells and murine B cells--which could not be detected after incubation of lymphocytes with healthy cells. The interaction was a direct one, not requiring the presence of accessory cells, but independent of cytotoxicity and TCR engagement. Electron microscopy disclosed 100-200 nm large gaps in the cell membranes of connected cells which separated viable and revealed astonishing outcome. While the lymphocytes were induced to proliferate in a long term fashion, the tumor cells underwent a temporary break in cell division. The in vitro results were confirmed in vivo using a murine acute lymphoblastic leukemia (ALL) model. The arrest of tumor proliferation resulted in a significant prolonged survival of challenged mice. CONCLUSIONS: The reported cell-cell contacts reveal new characteristics i.e. the enabling of cytosol flow between the cells including biological active proteins that influence the cell cycle and biological behaviour of the recipient cells. This adds a completely new aspect in tumor induced immunology.

Naïve rat NK cells control the onset of T cell response.

NK cell function in the rat is only defined in a rudimentary way due to missing tools for clear NK cell identification. The present study introduces the congenic LEW.BH-NKC rat strain which allows distinct detection of rat NK cells using commercial antibodies. LEW.BH-NKC rats were exposed in vivo to the porcine B cell line L23 by subcutaneous transfer of L23 cell suspension. We used Luciferase transgeneic L23 cells to follow the course of rejection by living imaging. L23 cells were rejected within five days after placement under the skin thus the rejection is mediated by innate immune responses in the first place. Indeed we found increased percentages of NK cells in the blood, spleen and in draining lymph nodes using flow cytometry methods. Surprisingly, we found as a consequence a decrease in proliferative T cell response in the draining lymph nodes. We identified NK cells as mediators of this regulation by in vitro performed mixed lymphocyte reactions. The remarkable feature was the naive state of NK cells exhibiting the regulative capacity. Furthermore, the regulation was not exclusively mediated by IL-10 as it has been reported before for influence of T cell response by activated NK cells but predominantly by TGF-ß. Interestingly, after initiation of the adaptive immune response, NK cells failed to take influence on the proliferation of T cells. We conclude that naive NK cells build up a threshold of activation impulse that T cells have to overcome.