Methods: The Phl p 5-transgenic mouse (Balb/c background) was generated by pronuclear injection integrating Phl p 5 fused to a leader peptide and a transmembrane domain under control of a CMV-promotor with a GFP-reporter. Splenocytes (2.5x106 per mouse), cell extracts containing mainly membrane proteins and recombinant (r) Phl p 5 [5µg+/- Al(OH)3 per mouse] were injected subcutaneously into naïve Balb/c mice (n=8 per group). The amount of Phl p 5 of splenocytes and cell extracts was semi-quantitatively determined via western blotting. Furthermore skin of Phl p 5-transgenic mice was grafted onto naïve Balb/c (n=13 in two independent experiments), a rejection model in allo-transplantation since skin is highly immunogenic and therefore readily rejected. Phl p 5-specific antibody response was determined in sera by ELISAs. For T-cell responses splenocyte-proliferation was assessed in vitro after stimulation with rPhl p 5.
Results: Surprisingly, a prompt rejection (within 8-10 days) was elicited, accompanied by a strong Phl p 5 specific antibody response including Phl p 5-specific IgE in wildtype Balb/c after skin rejection of Phl p 5-transgenic mice. Additionally to the skin grafted group, mice receiving splenocytes or rPhl p 5 plus adjuvant showed a comparable response in matters of Phl p 5-specific IgE- and IgG1-levels through the whole follow-up (week 1, 2, 3, 5, 7, 9, 11) suggesting an unusually strong immune response to cell or tissue-bound Phl p 5. Furthermore Phl p 5-specific IgG2a/2b/3, IgA and IgM were induced.
Besides in vitro splenocyte-proliferation assays showed Phl p 5 specific T-cell responses in all groups of mice that showed strong humoral responses.
Conclusions: The high immunogenicity of tissue-bound Phl p 5 may represent a new mode of rendering antigens highly immunogenic.