Presented at:
2007 American Transplant Congress
San Francisco, CA, May 5-9, 2007
Intragraft RNA Expression of FOXP3 Versus Proinflammatory Cytokines in Tolerant Recipients of HLA Mismatched Renal Allografts
Kawai T, Suthanthiran M, Sharma V, Ding R, Sykes M, Colvin RB, Cosimi AB, Sachs DH
Transplantation Unit, Massachusetts General Hospital, Boston, MA; Transplantation Biology Research Center, Massachusetts General Hospital, Charlestown, MA; Pathology, Massachusetts General Hospital, Boston, MA; Medicine, Cornell University, New York, NY; This Study Was Performed in Partnership with Immune Tolerance Network
BACKGROUND: Recent studies have demonstrated active recruitment of T regulatory cells (Treg) into a transplanted organ as an important mechanism of allograft tolerance. We have investigated intragraft expression of Foxp3 and proinflammatory cytokine genes in four tolerant recipients of HLA mismatched kidney allografts.
METHODS: Five subjects underwent combined kidney and bone marrow transplantation (CKBMT) from HLA mismatched donors. Renal allograft biopsies from tolerant recipients (TOL, 6 biopsies from 4 CKBMT recipients who have stable kidney function without immunosuppression), acute rejection (AR, 4 patients with conventional immunosuppression who developed histologically proven acute rejection) and normal donors (NL, n=5 ) were tested for mRNAs using pre-amplification enhanced kinetic quantitative PCR assays. mRNA copy numbers were normalized using 18sRNA copy numbers and log-transformed.
RESULTS: After transient mixed chimerism, complete withdrawal of immunosuppressive medication was achieved in four of five recipients with longest immunosuppression-free survival exceeding 3 years. In vitro assays showed donor specific immunological unresponsiveness, as well as involvement of CD4+CD25+Treg for the mechanisms of tolerance. Levels of intragraft mRNA for granzyme B (GB), perforin, interferon gamma, IP-10, CXCR3, CD3e, CD20 or CD25 were significantly lower in TOL compared to AR, while there was no significant difference in levels of GB, interferon gamma, CD20 and CD25 between TOL and NL. mRNA levels of Foxp3 were significantly higher in TOL (5.4±0.4) compared to NL (2.75±0.4), but significantly lower compared to AR(7.0±0.2). Up-regulation of Foxp3 without inflammatory responses in the tolerant subjects led to higher ratios of Foxp3 to GB mRNA levels in TOL(7.2±0.8) than in AR(4.2±0.4, P=0.01) or NL( 5.6±0.7, P=0.07).
CONCLUSIONS: Real-time PCR studies of intra-graft marker gene expression suggested the development of regulatory cells may be involved for the maintenance of tolerance after disappearance of chimerism.