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.

Disorders in regulatory T-cell (T(reg)) function can result in the breakdown of immunological self-tolerance. Thus, the identification of mechanisms controlling the activity of T(reg) is of great relevance. We used T(reg) from individuals carrying the C77G polymorphism as models to study the role of CD45 molecules in humans. C77G prevents splicing of CD45 exon A thereby leading to an aberrant expression pattern of CD45 isoforms in affected individuals. Resting and in vitro expanded/activated CD4(+)CD25(high)Foxp3(+) T(reg) from carriers of C77G strongly expressed CD45RA isoforms whereas these isoforms were almost absent in cells from individuals with wild-type CD45. C77G T(reg) showed diminished upregulation of activation markers, lower phosphorylation of p56(lck)(Y505) and a reduced proliferative potential when stimulated with anti-TcR or anti-TcR plus CD28 mAb suggesting decreased responsiveness to activating stimuli. In addition, the capacity to suppress proliferation of conventional CD4(+) T cells was impaired in C77G T(reg). Furthermore, microarray studies revealed distinct gene expression patterns in T(reg) from C77G carriers. These data suggest that the changes in CD45 isoform combination resulting from the C77G mutation alter the responsiveness of T(reg) to TcR-mediated signaling. Targeting CD45 isoform expression might be a useful approach to modulate T(reg) function.

Background T cell activation is regulated by the integration of positive and negative signals. A strategy to down‐regulate human immune responses to a porcine xenograft is the enhancement of negative costimulatory signals. The programmed death‐1 receptor (PD‐1) is one of the known negative regulators for T cell activation and we have previously shown that human in vitro T cell responses to porcine transfectants over‐expressing human PD‐Ligand 1 (PD‐L1) are reduced as compared to responses triggered by control cells. However, PD‐1 is also expressed on B cells and its role in B cell activation is not well understood so far. Thus, the present study focused on the effects of PD‐1/PD‐L1 targeting on the humoral immune response to xenoantigens. Methods In vitro activation of human B cells was induced by co‐culturing with porcine L23 cells (B cell line) genetically engineered to over‐express human PD‐L1 (L23‐PD‐L1 cells) or “mock” ‐transfected controls. In vivo immune responses to the two cell types were characterized in a rat model by immunization experiments. The anti‐pig antibody titer in the culture supernatant or the sera of rats was examined by binding to L23 cells and visualized by flow cytometry using FITC‐conjugated IgM and IgG. Results Stimulation of human peripheral blood mononuclear cells (hPBMC) with L23 cells resulted in up‐regulation of the early activation marker CD69 on CD19+ B cells. Furthermore, we observed T cell dependent proliferation of B cells and differentiation to antibody secreting cells in vitro. This was shown by the detection of anti‐pig antibodies in the culture supernatant of hPBMCs stimulated with xenoantigen after 7 days and the titer increased till day 12, with higher amounts of IgM than IgG antibodies. When the activation of B cells was induced by L23‐PD‐L1 cells, lower antibody titers were determined in comparison to B cell activation induced by L23 control cells. Moreover, the humoral immune response was reduced when a soluble PD‐L1 molecule was applied to hPBMCs stimulated with porcine cells. To further characterize the antibody responses to L23‐PD‐L1 and control cells in vivo, we analyzed the level of anti‐pig antibodies in rats after cell transplantation under the kidney capsule. In line with the in vitro data we found lower levels of antibodies in the serum of rats immunized with L23‐PD‐L1 cells than in rats receiving L23 control cells. Conclusion These data suggest that the antibody response to porcine xenoantigen is diminished in the presence of the inhibitory ligand PD‐L1. Application of PD‐L1 might be a strategy to prevent induction of anti‐graft antibody responses, which are particularly strong in the pig‐to‐primate combination and represent a significant immunologic barrier for clinical xenotransplantation.