The intragraft gene activation of markers reflecting T-cell-activation and -cytotoxicity analyzed by quantitative RT-PCR in renal transplantation.

T-cell activation is the key event in the development of acute allograft rejection and precedes clinically apparent organ damage. We have performed competitive RT-PCR to quantify the intragraft gene expression for T-cell associated cytokines (IL-2, IL-4, IL-7, IL-15), CTLA4 and cytotoxic lymphocyte specific molecules to test their potential as rejection markers and to further elucidate mechanisms involved in graft rejection. RNA was isolated from snap-frozen portions of core biopsies obtained for the evaluation of graft dysfunction in 34 adults and 8 children. Reverse transcription derived cDNA was coamplified with a known amount of a competitor (a mutated target gene fragment) and normalized for the house keeping gene GAPDH. IL-2, the principal T-cell growth factor and IL-4 were not detectable in any biopsy at the time of histologically apparent rejection. Transcripts of the novel cytokine IL-15 were found in all dysfunctioning grafts and in two donor kidneys prior to reperfusion. CTLA-4, expressed in activated T-cells after costimulation by CD28 was uniformly present post transplantation, but not in the two donor kidneys. Transcripts for IL-7 (p < 0.001), IL-15 (p < 0.0005), CTLA4 (p = 0.04), granzyme B (p < 0.00015) and perforin (p < 0.0003) showed a significant correlation to acute rejection episodes. Heightened gene expression declined rapidly after initiation of rejection treatment. Fas-ligand mRNA gene expression was upregulated in both acute and chronic rejections. While this study shows that competitive RT-PCR is a reliable diagnostic tool to detect acute rejection in renal core biopsies, a future challenge will be to identify molecular markers of evolving rejections utilizing RT-PCR in sequential samples of fine needle aspirations, urine and blood.