Between January 2010 and December 2020, 72 patients underwent KT with a CsA-based immunosuppressive regimen at the Hiroshima University Hospital. Of these, five were excluded from the study because of incomplete immune monitoring data caused by a limited volume of stored lymphocytes from donors for in vitro MLR assays. The remaining 67 patients (41 ABO-blood-type-compatible recipients [ABO-C] and 26 ABO-blood-type-incompatible recipients [ABO-I]) were enrolled in the study. A complete clinical history, including age, sex, primary disease, body mass index, human leukocyte antigen (HLA) mismatch, relationship, and dialysis period, was recorded at the time of transplantation.
Elderly recipients were defined as those aged ≥ 60 years (≥ 75th percentile; 49 non-elderly vs. 18 elderly patients). This study was conducted in accordance with the Declaration of Helsinki and was approved by the institutional review board of the Hiroshima University Hospital (No. Hi-77), and informed consent was obtained from all patients.
HLA typing and anti-HLA antibody testing
HLA (-A, -B, -C, -DRB1, and -DQB1) typing of donors and recipients was performed by xMAP R Technology of Luminex Corp., using polymerase chain reaction--sequence-specific oligonucleotide probes at high resolution (Wakunaga, Hiroshima, Japan). Anti-HLA antibodies in all recipients were analyzed before transplantation and monitored annually following transplantation. Serum samples were examined for IgG antibodies against HLA class I or II using methodologies such as WAKFlow (Wakunaga) or LABScreen Mixed (One Lambda Inc. Canoga CA). All positive evaluations were re-screened, and donor-specific anti-human leukocyte antigen antibodies (DSAs) were identified using the LABScreen Single Antigen (One Lambda). Mean fluorescence intensity values above 1,000 for DSAs against the two fields for HLA-A, -B, -C, -DRB1, and -DQB1 were considered positive.
Desensitization protocol and immunosuppressive regimen
Preoperative desensitization was performed in ABO-blood-type incompatible cases. Two weeks before transplantation, a single dose of rituximab (375 mg/m2 body surface area) was administered to the patients. Subsequently, they received CsA (target trough level: 80-100 ng/mL) and mycophenolate mofetil (MMF; 20 mg/kg/day) and underwent between zero and five plasmapheresis sessions to achieve at least a 16-fold preoperative reduction in anti-blood group isoagglutinin titers. The basic CsA-based immunosuppressive regimen following KT has been previously described 16.
Immune monitoring via in vitro mixed lymphocyte reaction assays
To evaluate the anti-donor immune reactivity of the patients, T cell responses to alloantigens were evaluated via MLR assays using an intracellular CFSE labeling technique. The detailed regimens and calculation of the stimulation indices (SIs) have been described previously 16. To evaluate the immune reactivity of recipients, CFSE-MLR assays were performed at 1, 2, and 3 weeks, after 1, 3, 6, and 12 months, and annually after KT. After analyzing the proliferation of CD4+ and CD8+ T cell subsets in response to anti-donor and comparing it with that in response to anti-third party stimuli in protocoled MLR, we categorized the immune status as hypo-, normo-, or hyperresponsive 30. Therapeutic adjustments for immunosuppressants were determined by tapering dosages in cases exhibiting anti-donor hyporesponsiveness in both T cell subsets and increasing them for anti-donor hyperresponsiveness.
Definitions and other laboratory data
Serum creatinine levels were monitored every day until postoperative week 2 and at least every other day until postoperative week 4. TCMR was defined as graft dysfunction as evidenced by elevated serum creatinine levels in the absence of vascular or urinary complications or infection. Vascular and urinary complications were identified using Doppler ultrasonography (US). The clinical suspicion of TCMR was supported by the protocoled MLR assay, which can rigorously monitor rejection 16. TCMR diagnosis was based on the Banff criteria in episode biopsies. Episodes of rejection were initially treated with either mini pulse (125-250 mg intravenous methylprednisolone (MP) for ≥ 2-3 days) or steroid pulse (500 mg intravenous MP for ≥ 3 days) according to the clinical severity of TCMR, with a gradual tapering of the dose and return to the previous oral triple-drug regimen.
The criteria for urinary tract infections (UTIs) were the presence of microbes at a concentration of > 104 CFU/mL of urine or > 103 CFU/mL after culture with clinical signs and symptoms and the use of antibacterial agents. Cytomegalovirus (CMV) antigenemia-positive was defined as the detection of ≥ 3/50,000 CMVpp65-positive cells. Clinical and laboratory data were extracted from the patient’ medical charts.
Statistical analyses were performed using the JMP version 16 (SAS Institute, Cary, NC, USA). Quantitative variables are expressed as mean ± standard deviation or median and range. Student’s t-test, Wilcoxon-Mann-Whitney test, chi-squared test, and Fischer’s exact test were used to compare variables between the two groups. Kaplan-Meier analysis was used to compare the time-to-event variables. The differences between the curves were examined using the log-rank test. Statistical significance was set at P < 0.05.