Hemodialysis Vintage, Black Ethnicity, and Pretransplantation Antidonor Cellular Immunity in Kidney Transplant Recipients
Joshua J. Augustine*,
Emilio D. Poggio,
Michael Clemente,
Mark I. Aeder,
Kenneth A. Bodziak*,
James A. Schulak,
Peter S. Heeger and
Donald E. Hricik*
* Division of Nephrology and Hypertension and Department of Surgery, University Hospitals Case Medical Center, and Departments of Nephrology and Hypertension; and Immunology, Cleveland Clinic, Cleveland, Ohio
Address correspondence to: Dr. Joshua Augustine, Division of Nephrology and Hypertension, Case Medical Center, 11100 Euclid Avenue, 1817 Mather, Cleveland, OH 44106. Phone: 216-844-8060; Fax: 216-844-5204; E-mail: joshua.augustine{at}uhhospitals.org
Received for publication October 10, 2006.
Accepted for publication February 12, 2007.
Prolonged exposure to dialysis before transplantation and blackethnicity are known risk factors for acute rejection and graftloss in kidney transplant recipients. Because the strength ofthe primed antidonor T cell repertoire before transplantationalso is associated with rejection and graft dysfunction, thisstudy sought to determine whether hemodialysis (HD) vintageand/or black ethnicity affected donor-directed T cell immunity.An enzyme-linked immunosorbent spot (ELISPOT) assay was usedto measure the frequency of peripheral T cells that expressedIFN- in response to donor stimulator cells before transplantationin 100 kidney recipients. Acute rejection occurred in 38% ofELISPOT (+) patients versus 14% of ELISPOT (–) patients(P = 0.008). The median (HD) vintage was 46 mo (0 to 125 mo)in ELISPOT (+) patients versus 24 mo (0 to 276 mo) in ELISPOT(–) patients (P = 0.009). Black recipients had a greatermedian HD vintage (55 versus 14 mo in nonblack recipients; P< 0.001). Black recipients with less HD exposure had a lowincidence of an ELISPOT (+) test, similar to nonblack recipients.Among variables examined, only HD vintage remained a significantpositive correlate with an ELISPOT (+) result (odds ratio peryear of HD 1.3; P = 0.003). These data suggest that the riskfor developing cross-reactive antidonor T cell immunity increaseswith longer HD vintage, providing an explanation for the previouslyobserved relationship between increased dialysis exposure andworse posttransplantation outcome. Longer HD vintage may alsoexplain the increased T cell alloreactivity that previouslywas observed in black kidney recipients.
Longer dialysis vintage (1–3) and black ethnicity (4)are clinical variables that are associated with inferior long-termoutcomes after kidney transplantation. T cell immunity, knownto play a prominent role in allograft rejection, may be linkedto both of these factors. Prolonged exposure to dialysis maylead to increased exposure to environmental antigens and effectorT cell formation. Increased cellular alloreactivity also hasbeen observed in black kidney transplant recipients. Kermanet al. (5) reported that, compared with white patients who wereawaiting transplantation, black patients exhibited higher Tlymphocyte helper:suppressor ratios and panel mixed lymphocytereactions. In addition, Poggio et al. (6) recently found a trendfor higher frequencies of IFN-–producing T cells in blackdialysis patients who were exposed to a panel of HLA antigens.
It is unclear whether black recipients have greater inherentT cell immunity or whether this is acquired after exposuresto environmental antigens. Longer waiting times on dialysishave been noted in black transplant candidates, with less timelyaccess to transplantation (7). Greater dialysis exposure hasbeen linked to a greater risk for rejection relative to preemptivetransplantation (2), although an underlying explanation forthis risk has not been apparent.
We hypothesized that prolonged hemodialysis (HD) vintage mayinfluence the strength of the primed cellular alloimmune repertoireand that this risk may be independent of black ethnicity. Toassess T cell alloimmunity, we used a highly sensitive enzyme-linkedimmunosorbent spot (ELISPOT) assay that quantifies the numberof IFN-–producing effector/memory T cells in human peripheralblood after exposure to donor cells (8). We previously demonstratedthat pre- and posttransplantation cellular immunity as measuredby the ELISPOT assay correlates with acute rejection (AR) andrenal functional impairment in all patients and specificallyin a cohort of black kidney transplant recipients (9–11).In the experience reported herein, we sought to analyze theimpact of HD vintage and black ethnicity on pretransplantationdonor-directed cellular immunity.
Patient Selection
Patients who received kidney transplants between January 2000and December 2003 were enrolled under the approved guidelinesof the Institutional Review Board for Human Studies at The UniversityHospitals of Cleveland. Patients provided informed consent beforetransplantation and were selected for ELISPOT immune monitoringon the basis of the availability of donor stimulator cells thatconsisted of donor splenocytes in deceased-donor (DD) transplantsor of donor peripheral blood mononuclear cells in living-donor(LD) transplants. All patients had pretransplantation ELISPOTtesting and received a kidney transplant either preemptively(n = 8) or after treatment with HD (n = 92). Recipients whowere enrolled in the immune monitoring study and received multipleorgan transplants (n = 12) or who were on peritoneal dialysisbefore transplantation (n = 8) were excluded from analysis.This cohort included a subset of black patients who were analyzedin a previous report (11).
Maintenance immunosuppression consisted of calcineurin inhibitortherapy in all patients (91 with tacrolimus, nine with cyclosporine).Sirolimus was used adjunctively in 62 patients, whereas mycophenolatemofetil therapy was given in the remaining 38. At the time ofenrollment, our protocol for black patients consisted of tacrolimus/sirolimuscombination, which was used in 93% of black patients. Alternatively,79% of nonblack patients received tacrolimus/mycophenolate mofetil.All patients were initiated on corticosteroids; 25 withdrewfrom steroids within 6 mo after transplantation. Induction therapywas given in select cases, primarily in patients with equivocallypositive cross-matches as determined by flow cytometry. Antithymocyteglobulin or basiliximab was used as an induction agent. An ELISPOTassay was performed using recipient cells that were collectedbefore immunosuppression and before kidney implantation. T cell–depleteddonor cells were used as stimulators in the assay. Choice andadjustment of immunosuppression was not influenced by the resultof the ELISPOT assay, which was not prospectively availableto the transplant clinicians.
Demographic variables were collected on patients at the timeof enrollment. HD vintage was recorded in months and representedtotal cumulative months on HD in patients who received nonprimarykidney transplants (n = 7). Renal biopsies were performed onpatients with suspected AR, and biopsy-proven AR was definedby a Banff IA or higher score (Banff ’97). Delayed graftfunction (DGF) was defined as the need for dialysis during thefirst week after transplantation. There was one graft loss duringthe first year in a patient who had positive testing by ELISPOTand had biopsy-proven AR within 1 mo of transplantation. Thispatient was included in the analysis.
HLA Typing and Alloantibody Determinations
Antigens that were encoded by HLA class I loci (A and B) wereidentified by the basic microlymphocytotoxicity assay usinglocal antisera. Class II alleles were determined by sequence-specificpriming and PCR. Pretransplantation panel reactive antibody(PRA) was determined by flow cytometry using Flow PRA beads(One Lambda, Canoga Park, CA) according to the manufacturer'srecommendations. We defined patients as highly sensitized whentheir final pretransplantation PRA was >60%.
ELISPOT Assays
IFN- ELISPOT assays were performed as described previously indetail (9). The resulting spots were counted with a Series 1Immunospot computer-assisted ELISPOT image analyzer (CellularTechnology, Cleveland, OH). Results were depicted as the meannumber of IFN- spots per 300,000 recipient peripheral bloodlymphocytes based on duplicate or triplicate measurements ina given assay. Based on previous analyses, a positive test waspredefined as a >25 spots per 300,000 peripheral blood lymphocytes.Control wells that assessed cytokine production by stimulatorsalone were included in all assays (<20 spots per 300,000),and detected spots in these control wells were subtracted fromthe total number of spots in wells in which responders and stimulatorswere mixed.
Statistical Analyses
Values are shown as mean ± SD, median (range), or percentage.Baseline demographic data between patient groups was analyzedusing t test for continuous variables and Pearson 2 test fordichotomous variables. A Mann-Whitney U test was used to compareHD vintage between groups and to compare the total number ofIFN- spots between groups, whereas 2 testing was used to compareELISPOT status between groups. Spearman correlations were usedto determine variables that were associated with HD vintage.Multivariable logistic regression analysis was used to determinefactors that were associated with AR and factors that were associatedwith an ELISPOT (+) test. Variables with P 0.10 in univariableanalyses were included in multivariable models. In addition,the variable "black ethnicity" was forced into the regressionanalysis for AR. Two-sided P < 0.05 was considered to indicatestatistical significance. All analyses were performed usingSPSS version 11.5 (SPSS, Chicago, IL).
Baseline demographic and transplant data are shown in Table 1.Patients were divided fairly evenly between black and nonblackpatients, with 53 black recipients in the cohort of 100 patients.The majority of transplants were performed using DD, and 81%of patients received no induction therapy. Sixteen percent werehighly sensitized, and 7% were recipients of a previous kidneytransplant.
We first looked at factors that were associated with biopsy-provenAR diagnosed in the first 12 mo after transplantation. The overallincidence of AR was 21%. Confirming known risk factors, univariableanalysis revealed associations between AR and both HLA mismatch(odds ratio [OR] per HLA mismatch 1.4; P = 0.04) and DGF (OR3.8; P = 0.012). In addition, there was a trend for more ARin DD transplant recipients (OR 2.5; P = 0.09). There was nocorrelation with age, gender, previous transplant, high PRA,induction therapy, or type of maintenance immunosuppression.Black recipients had a 25% incidence of AR compared with 17%in nonblack recipients, a nonsignificant trend but consistentwith previous reports (4,12). In addition, patients with ARhad a median HD vintage of 47 (8 to 120) versus 33 mo (0 to276 mo) in patients without AR (P = 0.06).
As in our previous work, patients with positive primed antidonorcellular immunity before transplantation had a higher incidenceof posttransplantation AR. The 12-mo incidence of AR was 38%in ELISPOT (+) patients versus 14% in ELISPOT (–) patients(P = 0.008; Figure 1). We used a multivariable logistic modelfor AR controlling for HLA, DGF, donor source (LD versus DD),black ethnicity, and HD vintage (Table 2). In this model, ELISPOT(+) status remained an independent correlate of AR (OR 4.6;P = 0.009). Addition of use of induction therapy and pretransplantationPRA resulted in a persistent association of ELISPOT (+) statuswith AR (OR 4.1; P = 0.02). In these models, the trend thatwas seen in univariate analyses for HD vintage in associationwith AR dropped out. This suggested a potential interactionbetween ELISPOT status and exposure to HD.
Table 2. Logistic regression analysis for 12-mo incidence of AR
We therefore next examined the relationship between IFN- ELISPOTresults and HD vintage. Median HD vintage for the entire cohortwas 36 mo (0 to 276 mo). HD vintage was significantly greaterin ELISPOT (+) patients with a median of 46 (0 to 125) versus24 mo (0 to 276 mo) in ELISPOT (–) patients (P = 0.009).Eight patients received a preemptive transplant, and of these,one (13%) was ELISPOT (+). In patients who were on HD <36mo (n = 48), 15% were ELISPOT (+). Conversely, in patients whowere on HD 36 mo (n = 52), 42% were ELISPOT (+) (P = 0.002;Figure 2).
Figure 2. Total number of IFN- ELISPOT in patients who were on hemodialysis (HD) <36 mo (left) and 36 mo (right). The dashed line represents the cutoff for an ELISPOT (+) test (25 spots). Percentages represent the percent ELISPOT (+) from each group.
We next examined the relationship among pretransplantation antidonorT cell immunity, HD vintage, and ethnicity (black versus nonblack).Median HD time for nonblack patients was 14 mo (0 to 276 mo),with one outlier in this group. Alternatively, median time forblack patients was 55 mo (6 to 136 mo) (P < 0.001). Therewere no preemptive transplants in the black cohort, and evenamong black recipients of LD kidneys, the median HD vintagewas 20 mo (6 to 84 mo).
Total median number of ELISPOTs and the percentage of ELISPOT(+) patients relative to the median HD vintage of 36 mo wereexamined for nonblack and black recipients (Figure 3). In thenonblack cohort, patients who were on HD 36 mo had a 47% incidenceof ELISPOT (+) testing, versus 17% in those who were on HD <36mo (P = 0.03). Similarly, in the black cohort, those who wereon HD 36 mo had a 40% incidence of ELISPOT (+) testing versus11% in those who were on HD <36 mo (P = 0.03). Therefore,HD vintage, rather than black ethnicity, was the primary riskfactor for heightened cellular alloimmunity measured at thetime of transplantation. Rates of AR trended in a similar mannerwhen examined comparing ethnic groups and dividing by the mediandialysis exposure. AR occurred in four (15%) of 27 of non-blackrecipients with a vintage <36 mo versus four (24%) of 17with a vintage 36 mo (NS). Black recipients with an HD vintage<36 mo had AR occur in one (6%) of 18 patients, versus 12(34%) of 35 patients who were on HD 36 mo (P = 0.02).
Figure 3. The percentage of patients with an ELISPOT (+) test divided by nonblack or black ethnicity (x axis) and by HD vintage <36 or 36 mo (y axis).
Finally, we examined the relationship between ELISPOT statusand other clinical variables and confirmed that post-ELISPOTimmunosuppressive therapy was similar between groups (Table 3).Among variables examined, only HD exposure had a positive correlationwith ELISPOT result. There was a trend for more DD recipientsin the ELISPOT (+) group, which disappeared after controllingfor HD vintage (data not shown). There was no correlation betweenHLA matching and ELISPOT result. Interestingly, we noted a negativecorrelation between antibody sensitization and cellular alloimmunityand found that only one of 16 highly sensitized patients (PRA>60%) was also ELISPOT (+). The association between an ELISPOT(+) result and HD vintage remained significant after controllingfor antibody sensitivity (OR per additional year on HD 1.3;P = 0.003).
Our data suggest that longer HD vintage augments the pretransplantationfrequency of activated alloreactive T cells and provide an explanationfor the association between dialysis vintage and renal allograftsurvival. Greater dialysis exposure, known to have a negativeimpact on patient survival after kidney transplantation (13,14),also correlates with AR and death-censored graft survival (1–3).It has been argued that dialysis may increase the risk for ARby correcting immune dysfunction related to the predialysisuremic state (2). However, dialysis also may be associated withgreater exposure to infectious and environmental antigens overtime, and such immunologic stimuli may lead to the generationof activated or memory T cells that are capable of cross-reactingwith alloantigens after transplantation (15,16).
Because previous reports have suggested greater T cell alloresponsesin black kidney transplant recipients, we sought to determinethe impact of black ethnicity on IFN- expression relative toHD vintage. When we stratified black and nonblack patients,we found a significant increase in HD vintage among black patientsbut low levels of IFN- expression in black patients who spent<3 yr on HD. These data suggest that dialysis-related environmentalstimuli, rather than inherent variability of the T cell response,may contribute to immunologic differences between ethnic groups.
One recent analysis of black European kidney transplant recipientsfound no increased rate of rejection compared with a white cohort(17). The black European patients who were analyzed seemed tohave greater resources than many black transplant candidates(18), and dialysis vintage was not different between ethnicgroups in this study (E. Thervet, Hospital Necker, Paris, France;personal communication, April 10, 2006). In the United States,it is likely that multiple variables influence inferior outcomesin black transplant recipients, including problems with posttransplantationaccess to care and prescription coverage (19). However, increasedHD vintage seems to convey a direct immunologic risk beforetransplantation and may further disadvantage black recipients.
The ELISPOT test of IFN- expression reflects donor-reactiveeffector/memory T cell activity and likely represents cross-reactivityafter previous antigenic stimulation (20). A recent analysisusing a panel of stimulator cells demonstrated increased IFN-frequencies against an increasing variety of HLA proteins withincreased dialysis vintage (15), thereby demonstrating cross-reactivityagainst multiple foreign antigens. Such a screening test usinga so-called "panel-reactive memory T cell" analysis may providea determination of overall T cell immunization. Alternatively,the donor-reactive ELISPOT test performed in this study maybe analogous to the final antibody cross-match by assessingprimed T cell reactivity specific to donor cells. These testsmay enhance our pretransplantation assessment of immunologicrisk, because donor reactive IFN- expression and its associationwith AR were independent of both PRA and HLA matching.
One limitation of this analysis is that we did not track potentialinfluences of T cell stimulation in these patients before transplantation.Potential sources of immunostimulation on HD include the dialysismembrane, synthetic vascular access, exposure to blood products,and recurrent infections (21). Prospective measurements of IFN-frequencies using a panel-reactive memory T cell assay in anHD population would be ideal to track the influence of suchfactors over time.
These data demonstrate for the first time that a measure ofcellular alloreactivity correlates positively with both longerHD vintage and immunologic injury after kidney transplantation.This experience offers a novel explanation for the inferiorimmunologic outcomes that previously were observed in patientswith longer dialysis vintage. Further efforts to shorten waitingtime on dialysis before transplantation, particularly in blackrecipients, may lead to improved outcomes and perhaps less disparateoutcomes between ethnic groups.
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Abstract
Introduction
in response to donor stimulator cells before transplantation in 100 kidney recipients. Acute rejection occurred in 38% of ELISPOT (+) patients versus 14% of ELISPOT (–) patients (P = 0.008). The median (HD) vintage was 46 mo (0 to 125 mo) in ELISPOT (+) patients versus 24 mo (0 to 276 mo) in ELISPOT (–) patients (P = 0.009). Black recipients had a greater median HD vintage (55 versus 14 mo in nonblack recipients; P < 0.001). Black recipients with less HD exposure had a low incidence of an ELISPOT (+) test, similar to nonblack recipients. Among variables examined, only HD vintage remained a significant positive correlate with an ELISPOT (+) result (odds ratio per year of HD 1.3; P = 0.003). These data suggest that the risk for developing cross-reactive antidonor T cell immunity increases with longer HD vintage, providing an explanation for the previously observed relationship between increased dialysis exposure and worse posttransplantation outcome. Longer HD vintage may also explain the increased T cell alloreactivity that previously was observed in black kidney recipients. 
2 test for dichotomous variables. A Mann-Whitney U test was used to compare HD vintage between groups and to compare the total number of IFN-
0.10 in univariable analyses were included in multivariable models. In addition, the variable "black ethnicity" was forced into the regression analysis for AR. Two-sided P < 0.05 was considered to indicate statistical significance. All analyses were performed using SPSS version 11.5 (SPSS, Chicago, IL). 
36 mo (n = 52), 42% were ELISPOT (+) (P = 0.002; Figure 2). 
