In Panel A, the serial creatinine levels in Patients 1, 2, 4, and 5 are indicated in yellow, and the withdrawal of immunosuppressive medications is shown as tapering bars above each creatinine curve. Arrows indicate the time at which allograft-biopsy specimens were obtained from these four patients. To convert the values for creatinine to micromoles per liter, multiply by 88.4. Panel B shows the recent protocol biopsy specimens from each of these four patients (obtained on days 1135, 1087, 731, and 666, respectively) (periodic acid–Schiff). The findings in all of the specimens were within normal limits, except for the specimen from Patient 4, which showed segmental glomerular basement membrane duplication and C4d deposition (see Fig. 1 in the Supplementary Appendix for representative glomeruli from protocol biopsy specimens).

Sequential cell-mediated lympholysis assays in each patient against the donor (black circles) and third-party persons (gray circles) showed specific unresponsiveness to the donor at most post-transplantation time points tested (Panel A). Sequential assays of mixed-lymphocyte reactions in each patient against the donor (dark-blue bars) and third-party persons (light-blue bars) showed specific unresponsiveness to the donor, as indicated by return of anti–third-party, but not antidonor, responses after discontinuation of immunosuppressive therapy (Panel B). Arrows indicate the time of complete discontinuation of immunosuppressive therapy. Panel C shows intragraft levels of mRNA in renal allografts. Total RNA was isolated from renal-allograft biopsy specimens and reverse transcribed to cDNA, and levels of mRNA were measured with the use of preamplification enhanced real-time quantitative polymerase-chain-reaction assays. A total of 23 biopsy specimens were examined for intragraft levels of FOXP3 mRNA, granzyme B mRNA, and housekeeping gene 18S ribosomal RNA (18S rRNA). Of the 23 biopsy specimens, 6 were obtained from four patients with stable renal-allograft function who were not receiving immunosuppressive therapy (IS) (stable IS-free group). Eight biopsy specimens were obtained from eight patients with stable renal-allograft function and normal protocol biopsy results; these patients were receiving maintenance immunosuppressive drug therapy comprising tacrolimus and mycophenolate mofetil (stable-with-IS group). Five biopsy specimens were obtained from five kidney donors (normal-kidney group), and four biopsy specimens were obtained from four patients with biopsy-confirmed acute rejection (acute-rejection group). The mRNA copies were normalized with the use of 18S rRNA copies and log-transformed. The log-transformed mean (±SE) ratio of FOXP3 mRNA copies to 18S rRNA copies was 5.48±0.31 in the stable IS-free group, 3.90 ±0.27 in the stable-with-IS group, 2.85±0.34 in the normal-kidney group, and 7.09±0.38 in the acute-rejection group. The log-transformed mean ratio of granzyme B to 18S rRNA was 5.12±0.69 in the stable IS-free group, 4.53±0.59 in the stable-with-IS group, 3.41±0.75 in the normal-kidney group, and 9.76±0.84 in the acute-rejection group. Pre denotes pretransplantation.