ELSEVIER Periodic Assessment of Indirect Allorecognition Pathways in Renal Transplant Recipients With Long-Term Well-Functioning Graft A. Iniotaki-Theodoraki, J. Boletis, I. Tsoutsos, J. Bocos, Gr. Vosnides, E. Samouilidou, A. Kostakis, and H. Choremi-Papadopoulou C ARE of renal transplant (RTx) recipients involves the reduction of immunosuppression to the lowest effec- tive immunosuppressive drug doses to avoid drug side effects and complications. However, this reduction may result in the activation of the mechanism(s) responsible for graft rejection. A major problem in renal transplantation is to identify parameters clinically useful for monitoring im- munosuppressive treatment. Several investigators support the concept that the indi- rect presentation of donor MHC peptides to recipient T cells seems to be an important pathway for initiation and/or amplification of the rejection response mainly at the late posttransplant period.‘,2 Humam T helper cell pathways have differential sensitivity to cyclosporine A (CyA) with the indirect allorecognition pathway being the most suscep- tible to suppression by CyA in vitro.3 In clinical transplan- tation depression of indirect allorecognition pathway in RTx recipients seems to represent adequate immunosup- pression for graft retention, and patients not exhibiting such suppression are at increased risk for graft rejection.4 This study investigates the possibility of whether periodic assessment of the indirect allorecognition pathway can be used for the evaluation of immunossuppressive status in RTx recipients with long-term well-functioning grafts. This approach may be applicable in clinical transplantation regarding the monitoring of immunosuppressive treatment. MATERIALS AND METHODS Patients Fourteen RTx (12 men and 2 women, aged 39.2 5 8 years) were studied. Ail patients received grafts from living-related donors. Seven patients had experienced eight acute rejection (AR) epi- sodes the first 60 days’ post-TX. Immunosuppressive Treatment Standard immunosuppression at initiation included methylpred- nisolone 20 to 30 mgiday. azathioprine 1.5 to 2 mg/kg BW, and CyA 5 to 7 mg/kg BW/d adjusted to blood levels measured by CyA- specific radioimmunoassay. Study Protocol The response of patients’ T cells to alloantigens following direct and indirect presentation, to diphtheria and/or Candida antigen 0041-1315/97/$17.00 PII SO041 -1345(96)00083-8 250 (indirect presentation pathway), and to mitogen phytohemaggluti- nin (PHA) was determined 14 to 18 months’ post-TX and at various times during a follow-up period of about 30 months. At the first determination all recipients had stable graft function (serum creatinine levels <2.2 mg/dL). The functional data were compared with blood CyA levels, CyA dose, and current serum creatinine levels. Twenty normal volunteers (nonimmunosuppressed) who were used as controls (NC) were tested the same way as were RTx recipients. T-Cell Functional Assays For the proliferation assays of T cells, 2.10’ peripheral blood mononuclear cells (PBMC) were placed in flat-bottomed well of a microtiter culture plate in complete medium RPM1 1640 (Gibco) and 10% pooled human plasma (Sigma) along with (a) no stimu- lation (medium background), (b) diphtheria and/or Candida anti- gen, 100 PLglmL (Merieux), (c) pool of irradiated (3000 rad) allogeneic PBMC from three unrelated healthy control volunteers, (d) the pool of irradiated allogeneic PBMC that had been fully depleted of antigen-presenting cells (macrophages and B cells) by plastic adherence and T lympho-kwik (One Lambda Inc, remaining CDT, and CD:> cells less than 0.2%, tested by flow cytometry)), and (e) PHA, 10 &mL (Sigma). [“HI-Thymidine (DuPont) was incor- porated the last 18 hours of the 5-day culture. PBMC response to a specific stimulant was considered to be positive if the mean cpm was more than 5 SDS above the mean cpm in the medium background well. RESULTS Patterns of Response in Functional Assays For each sample, one of three T-cell functional status was observed.4 Positive response to all stimuli indicates intact direct and indirect allorecognition pathway and is desig- nated as +/+. At the first determination, six patients exhibited T-cell functional status of +/+ (four had experi- enced AR episodes). All NC had positive response to all stimuli (pattern of +/+). In RTx recipients functional status From the Departments of Immunology (A.I.-T., E.S., H.C.-P.) and Nephrology (J.B., LT., Gr.V.) and the Transplantation Unit (J.B., A.K.), Laiko General Hospital, Athens, Greece. This work was supported by grant from the Hellenic Ministry of Health. Address reprint requests to A. Iniotaki-Theodoraki, 34 Davaki Street, Alimos, Athens, Greece. 0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 29, 250-251 (1997)