Eosinophil infiltrates, typical of MHC class II disparate acute skin graft rejection, are critically dependent on the availability of IL-4. IL-4 mice reject MHC class II disparate skin grafts by a pathway of rejection where neutrophils play a direct causal role.

β2-Microglobulin (β2m)-derived peptides are minor transplantation Ags in mice as β2m-positive skin grafts (β2m+/+) are rejected by genetically β2m-deficient recipient mice (β2m−/−). We studied the effector pathways responsible for the rejection induced by β2-microglobulin-derived minor transplantation Ags. The rejection of β2m+/+ skin grafts by naive β2m−/− mice was dependent on both CD4 and CD8 T cells as shown by administration of depleting mAbs. Experiments performed with β2m−/−CD8−/− double knockout mice grafted with a β2m+/+ MHC class I-deficient skin showed that sensitized CD4 T cells directed at β2m peptides-MHC class II complexes are sufficient to trigger rapid rejection. Rejection of β2m+/+ grafts was associated with the production of IL-5 in vitro, the expression of IL-4 and IL-5 mRNAs in the grafted tissue, and the presence within rejected grafts of a considerable eosinophil infiltrate. Blocking IL-4 and IL-5 in vivo and depleting eosinophils with an anti-CCR3 mAb prevented graft eosinophil infiltration and prolonged β2m+/+ skin graft survival. Lymphocytes from rejecting β2m−/− mice also displayed an increased production of IFN-γ after culture with β2m+/+ minor alloantigens. In vivo neutralization of IFN-γ inhibited skin graft rejection. Finally, β2m+/+ skin grafts harvested from B6lpr/lpr donor mice, which lack a functional Fas molecule, survived longer than wild-type β2m+/+ skin grafts, showing that Fas-Fas ligand interactions are involved in the rejection process. We conclude that IL-4- and IL-5-dependent eosinophilic rejection, IFN-γ-dependent mechanisms, and Fas-Fas ligand interactions are effector pathways in the acute rejection of minor transplantation Ags.

Laser-assisted microdissection has become a unique technique for an accurate gene-expression profiling analysis in human tissues. The introduction of this approach requires the development of a reliable, efficient, and reproducible procedure for tissue processing. We report a systematic evaluation of the different relevant steps required to obtain sufficient quantity and good quality RNA for reverse transcriptase-polymerase chain reaction when using frozen surgical pathologic tissues as starting material. We propose an optimized and very efficient method with respect to time and effort that can easily be put into practice in research laboratory as well as in any pathology laboratory.

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