The recombinant cat allergen rFel d 4 and its putative human homolog MUP (major urinary protein, referred herein as “rFel d 4 like lipocalin”), are compared in vitro for their effects on human monocyte-derived dendritic cells (DCs). We investigate antigen uptake, maturation marker expression, induction and production of Th1/Th2-determining cytokines during the immune response with different methods (immunofluorescence, FACS, ELISA). The results gained by this master thesis show that human DCs react differently to the exposure of the two homologous lipocalins rFel d 4 and rFel d 4 like lipocalin, according to their allergenic potential: Although the two lipocalins are highly similar, the cat allergen induces a Th2 immune response, which is typical for allergic reactions, whereas the putative human homologue triggers a Th1 response.
Confocal imaging of live DC preparations shows internalization into the same vesicles of immature DCs using recombinant lipocalins rFel d 4 and rFel d 4 like lipocalin labeled individually with different fluorescent dyes. Co-localisation studies with transferrin as a marker for endosomal pathway and lysotracker as a marker for lysosoms as well as with MHC class II for the antigen processing and presentation pathway were performed to track the fate of the lipocalins, which were revealed to be similar for both lipocalins.
We found significantly lower maturation marker expression (CD40, CD80, CD83, CD86, MHC class II) in human monocyte-derived DCs treated with rFel d 4 compared to cells treated with the endogenous non-allergenic rFel d 4 like lipocalin.
Culture supernatants of DCs were analyzed by ELISA for their content of IL-12 (p70), and co-culture of monocyte-derived DCs and allogeneic naive T-cells were examined for the expression of the cytokines IFN and IL-13. The results show that the cat allergen rFel d 4 induces a lower IL-12 (p70) production in human DCs compared to rFel d 4 like lipocalin and that T cells, which have been co-cultured with rFel d 4 like lipocalin-treated DCs, have a significantly lower production of the Th2 cytokine IL-13 and a higher production of Th1 cytokine IFN compared to T-cells, which have been co-cultured with rFel d 4 treaded DCs. Taken together the results indicate that the crosstalk of DCs with lipocalin antigens can direct the type of the induced immune response.