Abstract
Previous studies suggest that quantifying donor-reactive memory T cells prior to kidney transplantation by IFNγELISPOT can assist in assessing risk of posttransplant allograft injury. Herein we report an analysis of IFNγELISPOT results from the multicenter, Clinical Trials in Organ Transplantation-01 observational study of primary kidney transplant recipients treated with heterogeneous immunosuppression. Within the subset of 176 subjects with available IFNγELISPOT results, pretransplant IFNγELISPOT positivity surprisingly did not correlate with either the incidence of acute rejection (AR) or estimated glomerular filtration rate (eGFR) at 6- or 12-month. These unanticipated results prompted us to examine potential effect modifiers, including the use of T-cell-depleting, rabbit anti-thymocyte globulin (ATG). Within the no-ATG subset, IFNγELISPOTneg subjects had higher 6- and 12-month eGFRs than IFNγELISPOTpos subjects, independent of biopsy-proven AR, peak PRA, HLA mismatches, African American race, donor source, and recipient age or gender. In contrast, IFNγELISPOT status did not correlate with posttransplant eGFR in subjects given ATG. Our data confirm an association between pretransplant IFNγELISPOT positivity and lower posttransplant eGFR, but only in patients who do not receive ATG induction. Controlled studies are needed to test the hypothesis that ATG induction is preferentially beneficial to transplant candidates with high frequencies of donor-reactive memory T cells.
Introduction
The identification and use of reliable risk assessment tools to make clinical decisions prior to and after kidney transplantation has the potential to improve long-term outcomes. Current immunosuppression regimens are generally dictated by transplant center-specific protocols that take into consideration traditional clinical risk factors including recipient race, recipient and donor age and donor source, supplemented by laboratory assessments of humoral sensitization [i.e., panel reactive antibodies (PRA) and/or donor-specific antibodies (DSA)]. Such approaches are associated with lower rates of acute rejection (AR) and improvements in 1-year patient and allograft survival, but have not significantly prolonged the half-lives of kidney transplants (1). As a consequence, multiple research groups, including our own, have endeavored to identify and test laboratory-based, quantifiable biomarkers potentially capable of predicting allograft rejection and graft loss.
Studies performed in both animal and human transplant recipients indicate that donor-reactive T cell memory, independent of humoral sensitization, is a potent barrier to successful transplantation (2–6). In previous work, we showed that donor-reactive memory T cells producing interferon gamma (IFNγ) in peripheral blood could be quantified using an ELISPOT assay (6). We reported data indicating that the frequency of donor-reactive T cells that produce IFNγ in ELISPOT assays (IFNγELISPOT) prior to kidney transplantation correlated with an elevated risk of posttransplant AR and lower 12-month glomerular filtration rate (GFR) (7, 8). Several other single center analyses have reported similar findings (9–13). Importantly, a multicenter analysis of the IFNγELISPOT assay as a pretransplant risk assessment tool has not been previously reported and was the goal of the current study.
The Clinical Trials in Organ Transplantation - Protocol 01 (CTOT-01) trial is a prospective, multicenter observational study designed to determine the diagnostic and predictive utility of a panel of candidate noninvasive biomarkers for transplant outcomes in primary kidney allograft recipients (14). As part of the CTOT-01 protocol we performed IFNγELISPOT assays before and after transplantation, stimulating recipient peripheral blood mononuclear cells (PBMC) with donor-derived cells to determine the frequency of donor-reactive memory cells producing IFNγ. Herein we report analyses correlating the pretransplant IFNγELISPOT results with posttransplant outcomes in this multicenter cohort.
METHODS
Study design and oversight
This prospective multicenter observational trial enrolled 280 adult and pediatric recipients of primary living or deceased donor kidney transplants performed between 2006 and 2009. Drs. Heeger and Hricik led the CTOT-01 protocol development team. Details of the trial design and oversight have been previously outlined (14). Each site participated under the auspices of its Institutional Review Board. An independent, NIAID-appointed Data Safety Monitoring Board was responsible for periodic safety review.
This report includes analysis of 176 of the 280 subjects for whom pretransplant IFNγELISPOT testing was available. The assay results were unavailable in 104 enrollees, predominantly because donor cells were not available and less often because of technical failure of the assay resulting from inadequate or non-viable cells.
Subjects
Adult and pediatric subjects undergoing a primary kidney transplant and having a negative flow cytometry crossmatch at the time of transplantation were eligible for enrollment. Plans for multi-organ transplantation and/or clinically significant liver disease were exclusion criteria.
Endpoints
The overall objective of CTOT-01 was to determine the relationships between results of immune assays and a composite primary endpoint (clinically evident or subclinical cellular AR of Banff grade IA or higher, increase in Banff chronic sum score> 2, increase in interstitial fibrosis > 15%, graft loss, or death at 6 months after transplant) and/or a change in renal function [>30% decrease in estimated GFR (eGFR)] between months 6 and 24 after transplant (14). In pre-specified secondary analyses we correlated the results of each assay with individual components of the composite endpoint (see Statistical Methods). A diagnosis of antibody-mediated rejection (AMR) was defined by histological criteria and/or C4d staining with or without a DSA as defined in the Banff criteria.
Interventions
Immunosuppression was not standardized in this observational study. Doses and levels of immunosuppressive drugs were defined and maintained as per local practice. Blood levels of calcineurin inhibitors were not collected or analyzed. The choice of induction therapy was left to the discretion of the local investigator. Allograft protocol biopsies were obtained at implantation and 6 months after transplantation. “For-cause” biopsies were obtained at the discretion of the treating physician.
Blood and urine samples were obtained prior to transplantation, on day 3, weeks 1–4, and months 2–6, 12 and 24 after transplant and serum creatinine measurements performed at 3, 6, 12 and 24 months (14). Protocol biopsies were read centrally (by IWG, University of Manitoba). Clinically indicated biopsies were read locally for clinical management. Tissue from the same biopsy was submitted to and read by the central CTOT-01 pathology laboratory. We defined biopsy-proven AR as Banff grade >1A. AR episodes were treated according to local practice.
Laboratory studies
IFNγELISPOT
Blood was collected in heparinized green top tubes and PBMC were isolated by Ficoll separation at each site within 6 hours of collection, and frozen using a standard operating procedure (15). Blood samples were obtained from living donors and processed similarly. PBMCs or spleen cells obtained from deceased donors were sent to the Mount Sinai core laboratory where they were processed and frozen.
Primary allostimulator B cell lines were expanded from each donor sample by culturing enriched B cells with CD40L transfected fibroblasts plus IL-4 as previously described (16). All B cell lines were phenotyped for HLA class II, CD40, CD80/86 and tested for their ability to stimulate allogeneic T cells as a quality assurance strategy. Only B cells that passed quality assurance standards were employed.
IFNγELISPOT assays were performed as previously described in detail (2, 15). Recipient PBMC (300,000 per well) were stimulated against donor and randomly chosen third party stimulators (100,000 per well) in triplicate. The resulting spots were counted with an Immunospot computer-assisted ELISPOT image analyzer (Cellular Technology, Cleveland, OH). Results were depicted as the mean number of IFNγ spots per 300,000 recipient peripheral blood lymphocytes based on duplicate or triplicate measurements in a given assay. A positive test was defined as more than 25 spots per 300,000 peripheral blood lymphocytes (see Statistical Methods). Control wells that assessed cytokine production by stimulators alone were included in all assays and detected spots in these assays were subtracted from the total number of spots in wells in which responders and stimulators were mixed. Donor-stimulated IFNγELISPOTS were performed pretransplant and when samples were available, repeated at 2 weeks, and 1, 3, 6, and 12 months posttransplant.
Serum Creatinine and eGFR
Serum creatinine levels were assayed centrally (Cleveland Clinic) using an IDMS-traceable analyzer (Hitachi Module D/P, Indianapolis, IN). Missing central laboratory sample results (46/404, 11.3%) were imputed from local creatinine values (Pearson’s correlation=0.92 between local and central values). A serum creatinine value of 7 mg/dl was imputed for graft loss. eGFR was calculated at 6 and 12 months post-transplant by the modified 4-variable MDRD equation for adults (17) and by the modified Schwartz equation for subjects younger than 18 years old (18).
Statistical Methods
Pre-specified secondary analyses of CTOT-01 were designed to assess the impact of confounding clinical factors and results of immune assays, including the IFNγELISPOT, on the primary endpoint and each of its components, including AR within the first 6 months and eGFR throughout the study. Receiver Operator Curve analyses indicated that the optimal cut point for an association between pretransplant IFNγELISPOTs and AR was 27 cells/300,000, and for eGFR was 16/300,000 PBMCs (data not shown), confirming our pre-specified threshold of 25/300,000 PBMC to define a positive test.
Data are summarized using descriptive statistics for categorical (counts/percentages) and continuous (mean and standard deviations) variables. Univariate analyses were performed using Chi Square or Fisher exact test for categorical variables and t-tests or analysis of variance (ANOVA) for continuous variables. A Kruskal-Wallis non-parametric test was used to compare variables that were not normally distributed. Linear regression was used to assess the independent associations of IFNγELISPOTs and other clinical variables on eGFR. Logistic regression was used to assess the independent associations of IFNγELISPOTs and other clinical variables on AR. A p value < 0.05 was considered to be statistically significant.
RESULTS
We initially compared the clinical characteristics of the 176 subjects with IFNγELISPOT results to those of the entire 280 CTOT-01 cohort (Table 1, left side). We observed no significant differences in age, gender, HLA mismatch, or any of the other shown variables indicating no selection bias for the IFNγELISPOT subset. Within this cohort, induction antibody therapy consisted of ATG in 85 subjects (48%), anti-IL-2R antibodies in 66 (38%) and no antibody induction in 25 (14%). As previously reported (14), maintenance immunosuppression was heterogeneous, but 84% of subjects received calcineurin inhibitor-based immunosuppression at the time of initial hospital discharge.
Table 1.
Clinical characteristics of study subjects versus all CTOT-01 enrollees (center side) and study subjects who received rabbit anti-thymocyte globulin (ATG) versus those who did not receive ATG (right side).
| Characteristics | All CTOT-01 Enrollees (N=280) | Subjects w/Pre-Trans ELISPOT (N=176) | Subjects w/Pre-Trans ELISPOT | ||
|---|---|---|---|---|---|
| ATG Subjects (N=85) | No-ATG Subjects (N=91) | P value | |||
| Donor Age (years) | |||||
| Mean (SD) | 39.8 (13.02) | 40.0 (12.93) | 41.5 (12.97) | 38.6 (12.81) | |
| Donor Gender | |||||
| Male | 134 (47.9) | 85 (48.3) | 42 (49.4) | 43 (47.3) | |
| Female | 146 (52.1) | 91 (51.7) | 43 (50.6) | 48 (52.7) | |
| Donor Type | |||||
| Deceased | 88 (31.4) | 56 (31.8) | 43 (50.6) | 13 (14.3) | <0.01 |
| Living | 192 (68.6) | 120 (68.2) | 42 (49.4) | 78 (85.7) | |
| Recipient Age (years) | |||||
| Mean (SD) | 43.5 (17.57) | 46.6 (15.59) | 50.1 (12.60) | 43.4 (17.37) | <0.01 |
| Recipient Gender | |||||
| Male | 168 (60.0) | 105 (59.7) | 59 (69.4) | 46 (50.5) | |
| Female | 112 (40.0) | 71 (40.3) | 26 (30.6) | 45 (49.5) | |
| Recipient Race | |||||
| Black or African American | 80 (28.6) | 55 (31.3) | 38 (44.7) | 17 (18.7) | <0.01 |
| Other Race | 199 (71.1) | 120 (68.2) | 47 (55.3) | 73 (80.2) | |
| Unknown or Not Reported | 1 ( 0.4) | 1 ( 0.6) | 0 | 1 ( 1.1) | |
| Recipient Age Group | |||||
| Adult | 240 (85.7) | 164 (93.2) | 83 (97.6) | 81 (89.0) | <0.01 |
| Pediatric | 40 (14.3) | 12 ( 6.8) | 2 ( 2.4) | 10 (11.0) | |
| Peak PRA | |||||
| Mean (SD) | 12.9 (24.58) | 10.2 (20.27) | 11.4 (23.75) | 9.0 (16.43) | |
| Number of HLA Mismatches | |||||
| Mean (SD) | 3.5 (1.81) | 3.5 (1.81) | 3.9 (1.83) | 3.1 (1.71) | <0.05 |
| Induction Therapy | |||||
| Yes | 233 (83.2) | 151 (85.8) | 85 ( 100) | 66 (72.5) | <0.01 |
| No | 47 (16.8) | 25 (14.2) | 0 | 25 (27.5) | |
| DeNovo DSA | |||||
| Yes | 11 ( 3.9) | 4 ( 2.3) | 1 ( 1.2) | 3 ( 3.3) | |
| No | 269 (96.1) | 172 (97.7) | 84 (98.8) | 88 (96.7) | |
We tested the strength of the correlations between a positive pretransplant IFNγELISPOT result with a) the predefined composite primary endpoint and b) the cumulative incidence of posttransplant AR and c) posttransplant eGFR at 6, 12 and 24 months. These multivariate analyses, performed on the entire study population, unexpectedly revealed no statistically significant relationships between pretransplant IFNγELISPOTs and either posttransplant AR or GFR. Non-significant trends suggesting relationships between pretransplant IFNγELISPOTs and 12-mo eGFR in this heterogeneously treated population raised the possibility that the previously observed correlation between a positive pre-transplant IFNγELISPOT and decreased eGFR by multiple research groups (3, 8–13) could have been influenced by various immunosuppressive medications, including the use of T-cell-depleting rabbit anti-thymocyte globulin (ATG). This concept is supported by documentation that ATG induction reduces the numbers of circulating CD4 and CD8 T cells while preserving regulatory T cells (19) and that ATG induction is linked to improved kidney transplant function/survival (20). Moreover, in a previously reported retrospective analysis of an independent, observational cohort, we observed that, among subjects who were IFNγELISPOTpos prior to transplant, those who received ATG induction had higher eGFRs at 12 months posttransplant than those treated with non-T-cell-depleting anti-interleukin-2 receptor (anti-IL-2R) antibodies (21).
To examine whether ATG induction influenced the relationship between IFNγELISPOT positivity and graft function in the CTOT-01 cohort, we performed post-hoc analyses after categorizing subjects based on induction therapy. Because the numbers of subjects treated with anti-IL-2R antibodies (n=66) and those who received no induction therapy (n=25) were relative low and anti-IL-2R mAb is non-T cell depleting we combined those treated with anti-IL-2R and those who received no induction into a single, no-ATG group and compared them to subjects who received ATG (ATG group), so as to optimize power for detecting differences. Importantly, we found no significant differences in either eGFR or the cumulative incidence of AR at any measured time point posttransplant (data not shown) between those treated with anti-IL-2R mAb and those who received no induction, supporting the combinatorial approach.
Clinical characteristics of the subjects, stratified by ATG vs. no-ATG (Table 1, right side) showed that the subjects in the ATG group were older, more often African American, and more often recipients of deceased donor transplants and of kidneys with more HLA mismatches.
The percentages of IFNγELISPOTpos subjects in the ATG group (44%) versus the no-ATG group (38%) were not different. Similarly, the mean absolute number of pretransplant IFNγELISPOTs produced by donor-reactive T cells in the ATG group (46.5 + 64.2) did not differ from that of the no-ATG group (51.9 + 137.2).
IFNγELISPOT positivity is associated with AR in subjects receiving ATG
Banff ≥1A rejection within the first 6 mo posttransplant occurred in 15/176 (8.5%) study subjects (Figure 1). AR occurred more frequently in ATG-treated subjects who were pretransplant IFNγELISPOT+ (7/37, 18.9%) than in ATG-treated subjects who were pretransplant IFNγELISPOTneg (2/48, 4.2%; p=0.037). No relationship was observed between IFNγELISPOT status and acute cellular rejection in the no-ATG group, but the low rates of rejection in these subjects (6/91, 6.6%) limit our ability to reach definitive conclusions. Pretransplant IFNγELISPOT status did not correlate with Banff scores on the 6-month protocol biopsies (data not shown).
Figure 1.
Incidence of acute rejection in the first 6 months posttransplant based on IFNγELISPOT positivity in all subjects (left panel), subjects treated with rabbit anti-lymphocyte globulin (ATG) (middle panel), and those not treated with ATG (right panel).
IFNγELISPOT status correlates with 6- and 12-month eGFR only in the no- ATG group
In the no-ATG subjects, pretransplant IFNγELISPOTneg subjects had higher eGFRs than IFNγELISPOTpos subjects (Figure 2) at 6 months (67.0 + 20.5 vs 57.2 + 17.8 ml/min; p<0.05) and 12 months (68.9 + 21.4 vs 59.0 + 18.8 ml/min; p<0.05). We observed similar, albeit non-significant trends of higher 12-mo eGFRs in the IFNγELISPOTneg vs. IFNγELISPOTpos subjects within the relatively small subsets treated with anti-IL-2R mAb (IFNγELISPOTneg : 67.42±21.87 ml/min, n=36 vs. IFNγELISPOTpos :58.7 ±20.75 ml/min, n=23, p=0.12, data not shown) or not given any induction (IFNγELISPOTneg: 72.72±20.60, n=14 vs. IFNγELISPOTpos: 59.82±15.06, n=8, p=0.17, data not shown). In contrast, within the ATG group IFNγELISPOT status did not correlate with differences eGFRs at either time point (Figure 2).
Figure 2.
Estimated glomerular filtration rates (eGFR) at 6 months (upper panels) and 12 months (lower panels) posttransplant based on IFNγELISPOT positivity in all subjects (left panels), subjects treated with rabbit anti-lymphocyte globulin (ATG) (middle panels), and those not treated with rATG (right panels).
We next performed multivariable linear regression analyses (Table 2) which confirmed an association between pretransplant IFNγELISPOTpos status and lower eGFR at 6- and 12-months only in the no-ATG group, independent of peak PRA, number of HLA mismatches, African American race, recipient age, recipient gender, and donor source. As expected, AR was associated with a lower eGFR in the no-ATG group (Table 2). Adding AR into the statistical model did not affect the relationship between a positive pretransplant IFNγELISPOT and lower posttransplant eGFR (IFNγELISPOT status correlates with lower eGFR independent of AR). Therefore, IFNγELISPOT status correlates with lower eGFR independent of AR within the no-ATG group.
Table 2.
Logistic regression coefficients for modeling estimated glomerular filtration rate at 6 and 12 months posttransplant in patients who did NOT receive anti-thymocyte globulin
| Parameter Estimates and tests-Full Model | |||||
|---|---|---|---|---|---|
| Month | Number of Obs. | Model Predictors | Coefficient (95% CI) | P- value | R2 |
| 6 | 88 | Pre-Transplant Donor Specific ELISPOT (+) | −9.84 (−18.29, −1.39) | 0.0231 | 0.0586 |
| 12 | 81 | Pre-Transplant Donor Specific ELISPOT (+) | −9.18 (−18.20, −0.16) | 0.0461 | 0.1287 |
| 12 | 81 | Pre-Transplant Donor Specific ELISPOT (+) | −9.97 (−19.01, −0.93) | 0.0311 | 0.1215 |
| AR in First 12 Months (Yes) | −18.15 (−32.88, −3.43) | 0.0164 | |||
Other variables tested that were not significant: HLA Mismatches, Peak PRA, Donor Type, AR in first 6months (6 Month GFR analyses), African American Race, Sex, Age, Low Risk
ATG was administered in this observational trial based on local practice and perceived clinical risk by each site investigator. While clinical risk assessments vary among sites, we reasoned that it was possible that clinical risk for posttransplant injury confounded the observed relationship between IFNγELISPOT positivity and lower eGFR independent of ATG use. In an effort to address this, we performed an additional post hoc analysis in which we stratified subjects as: a) “low risk,” defined as recipients of living donor allografts over the age of 50 and with negative DSA or “higher risk,” defined as subjects not meeting these criteria. Notably, in both groups use of ATG was equivalent (44% in low risk, 50% in high risk; p=ns).
The analysis revealed no correlation between pretransplant IFNγELISPOT status and either 6-month eGFR or incidence of AR, regardless of risk status (p > 0.2 for all analyses, data not shown), consistent with the interpretation that ATG induction modified the relationship between IFNγELISPOT positivity and lower posttransplant eGFR.
In further support of this conclusion, an analysis of anti-donor responses during the first 6 months posttransplant revealed significantly lower absolute numbers of IFNγELISPOTs in subjects treated with ATG compared to the no-ATG group (Figure 3). This finding supports the concept that ATG depletes donor-reactive T cells (regardless of the magnitude of the pretransplant IFNγELISPOT), an effect that is hypothesized to positively impact graft function (19, 20). We speculate that this ATG-induced depletion of anti-donor T cell immunity may protect the recipient from donor-reactive, memory T cell-mediated, allograft injury, and this modifies the predicted relationship between pretransplant IFNγELISPOT positivity and lower eGFR (as observed in the no-ATG group).
Figure 3.
Frequency of donor-reactive IFNγELISPOTs measured before and after transplantation in subjects treated with rabbit anti-lymphocyte globulin (rATG) (open circles, means depicted as blue bars) versus no rATG (closed circles, means depicted as red bars). + p < 0.01 at 1, 2 and 6 months posttransplant. In order to illustrate the changing mean ELISPOT values over time these values were truncated to 150 and are shown in red. The 27 values above 150 ranged from 155 to 1040.
Discussion
The results of this prospective, observational multicenter study add to the evidence that transplant measurements of donor-reactive memory T cells by IFNγELISPOT testing can inform clinicians regarding the risk of posttransplant kidney allograft function. While pretransplant donor-reactive IFNγELISPOT positivity did not correlate with risks of AR or worsened GFR in the entire heterogeneous CTOT01 cohort, our analyses indicate that positive test results are informative within the subset of subjects who did not receive ATG induction therapy. In the absence of ATG induction, IFNγELISPOTneg individuals had significantly higher eGFRs at 6- and 12-mo posttransplant versus those who were IFNγELISPOTpos, independent of known clinical risk factors associated with lower posttransplant GFR, including a previous episode of AR (Table 2). It is possible that pretransplant ELISPOT status is predictive of ongoing immunologic injury that negatively influences eGFR even in the absence of overt AR. In contrast, in subjects given ATG induction, we did not observe correlations between IFNγELISPOT status and posttransplant eGFRs. Together with: a) our demonstrated, depleting effects of ATG on donor-reactive T cells during the first 6 months posttransplant (Figure 3); b) previously reported results by our group (21); and c) those reported by Bestard et al (9) in which pretransplant IFNγELISPOT status of 60 ATG-treated subjects did not correlate with eGFR at 6 or 12 months, our new data raise the possibility that ATG induction therapy modifies the relationship between IFNγELISPOT positivity and posttransplant outcome. We speculate that ATG treatment sufficiently depletes donor-reactive memory T cells with or without facilitating Treg expansion (19) to mitigate the putative detrimental effects of anti-donor memory on graft function. A prospective study in which IFNγELISPOT positive allograft recipients are randomized to ATG or no-ATG would be required to test this hypothesis.
Although we showed no relationship between IFNγELISPOT status and eGFR in patients receiving ATG, we did observe that ATG-treated subjects who were IFNγELISPOTpos had a higher risk of posttransplant AR, confirming and extending results from our previous work and from studies performed by other groups. T cell-depleting induction therapy has been shown to favor the emergence of memory T cells when they are present prior pretransplant, and that these expanding T memory cells have the potential to mediate AR (22, 23). It is possible that the positive association between a positive pretransplant IFNγELISPOTpos status and posttransplant AR in ATG treated individuals was influenced by homeostatic re-expansion of donor-reactive memory T cells in our study, although we did not specifically obtain samples to assess this hypothesis. Whereas we did not observe a correlation between pretransplant IFNγELISPOT positivity and posttransplant AR in the no-ATG induction group, only 6/91 subjects developed AR over the initial 6 months in this subset, potentially indicative of a type II error.
In conclusion, results of the CTOT-01 multicenter study support the conclusion that pretransplant IFNγELISPOT testing can assess immune risk in kidney transplant recipients but also strongly suggest that ATG induction modifies this association. Additional randomized trials are needed to determine whether the IFNγELISPOT can be used to guide immunosuppression use, and, importantly, to identify patients who may benefit from T cell-depleting induction therapy.
Abbreviations
- AMR
antibody-mediated rejection
- AR
acute rejection
- CTOT
Clinical trials in Organ Transplantation
- eGFR
estimated glomerular filtration rate
- ELISPOT
enzyme linked immunosorbent spot assay
- DSA
Donor-specific antibodies
- PRA
panel reactive antibodies
Footnotes
Disclosure
The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.
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