Trends in immune function assay (ImmuKnow; Cylexä) results in the first year post-transplant and relationship to BK virus infection

2565 Nephrol Dial Transplant (2012) 27: 2565 2570 doi: 10.1093/ndt/gfr675 Advance Access publication 13 December 2011 Trends in immune function assay (ImmuKnow; Cylexä) results in the first year post-transplant and relationship to BK virus infection Jane Gralla 1,2, *, Janna Huskey 3, * and Alexander C. Wiseman 1,3 1 Transplant Center, University of Colorado Denver, Aurora, CO, USA, 2 Department of Pediatrics, University of Colorado Denver, Aurora, CO, USA and 3 Division of Renal Diseases and Hypertension, University of Colorado Denver, Aurora, CO, USA Correspondence and offprint requests to: Alexander C. Wiseman; E-mail: Alexander.wiseman@ucdenver.edu *These authors contributed equally to this work. Abstract Background. The ImmuKnow assay is a functional T- cell assay (TCA) that may quantify cellular immune responsiveness following renal transplantation. Using a standard protocol of TCA sampling in the first year post-transplant, we examined changes in TCA values over time and tested for an association between TCA and BK virus (BKV) infection as a marker of overimmunosuppression. Methods. We performed a single-center retrospective analysis of 897 TCA results in 414 renal transplant recipients obtained at 0 (N ¼ 122), 1 (N ¼ 316), 6 (N ¼ 258) and 12 (N ¼ 201) months post-transplant from May 2005 to July 2009 with concurrent urine and blood BKV polymerase chain reaction measurements. Results. Nearly 40% of patients experienced a decrease in TCA of >150 ng/ml from 1 to 6 months (mean 466 356 ng/ml, P < 0.0001) and remained stable from 6 to 12 months (mean 357 versus 370 ng/ml, P ¼ 0.33). Neither a change in TCA of >150 ng/ml nor a TCA value of 225 ng/ml were associated with a diagnosis of BKV infection at 1 or 6 months, while TCA 225 ng/ml was associated with BKV infection at 12 months (P ¼ 0.005). Conclusions. A reduction in TCA from 1 to 6 months posttransplant is common and is not associated with conditions of over-immunosuppression, rendering the interpretation of changes in TCA during this time period difficult. BKV infection is associated with low TCA values at 12 months, suggesting that patients with low TCA values after 6 months may benefit from potential tailoring of immunosuppression or more aggressive monitoring to prevent subsequent BKV infection. Keywords: cellular immunity; immune monitoring; kidney transplantation Introduction Kidney transplantation has enjoyed significant advances with respect to graft and patient survival over time [1]. However, allograft rejection and opportunistic infections (OIs) remain important contributors to graft survival, morbidity and mortality [2 4]. Maintaining a fine balance between over- and under-immunosuppression in order to minimize these complications remains an empiric and challenging endeavor. Additional non-invasive tools are Ó The Author 2011. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

2566 J. Gralla et al. needed beyond therapeutic drug monitoring to optimize immunosuppression and avoid untoward events [5, 6]. The ImmuKnow assay (Cylex) was approved by the US Food and Drug Administration (FDA) in 2002 as a means of measuring cell-mediated immunity in immunocompromised individuals [7]. This T-cell assay (TCA) quantifies the amount of intracellular ATP that is released from CD41 T cells in response to a non-specific mitogenic stimulus. Since the introduction of this test in 2002, several small studies have utilized this test to identify individuals at risk for both opportunistic and non-ois [8 14]. We recently published findings of a large retrospective analysis describing the lack of utility of single time point TCA testing in the prediction of rejection or OI [15]. As yet unanswered is the natural history of TCA values in kidney transplants recipients over time and whether changes in TCA values may be more predictive of OI than single time point assessments. In an effort to determine the natural history of changes in TCA results in individuals within the first transplant year, we performed a large retrospective analysis of TCA results obtained by protocol screening to identify trends of TCA results over time. Additionally, we correlated both the absolute TCA values and the changes in TCA values over time to the presence or absence of BK virus (BKV) re-activation as a means to analyze the ImmuKnow assay in its ability to predict a state of over-immunosuppression. We hypothesized that significant reductions in TCA values over time will be associated with the development of BKV infection. Materials and methods We performed a single-center retrospective analysis of protocol-driven ImmuKnow Assay results obtained in renal transplant recipients from May 2005 to July 2009. Individuals that were 18 years of age and had undergone renal transplantation or simultaneous pancreas kidney transplantation were included in the study if they had at least one qualifying ImmuKnow Assay drawn during the study period at pre-specified protocol time points. Baseline immunosuppression primarily consisted of prednisone, a calcineurin inhibitor and an anti-proliferative agent. Induction therapy was used in patients with at least one of the following characteristics: (i) repeat transplant, (ii) simultaneous pancreas kidney transplant, (iii) African- American race and (iv) panel-reactive antibody 20 %. ImmuKnow assays (T-cell assay, TCA) were drawn for protocol screening at 0, 1, 6 and 12 months post-transplant. The tests were performed at the University of Colorado HLA Laboratory as previously described [7]. Whole blood was incubated overnight with phytohemagglutinin. The CD4 1 cells were then selected using paramagnetic particles coated with monoclonal CD4 1 antibodies and lysed to release ATP. They were then processed through a luminometer to calculate the numeric result [7]. The mean values were analyzed at Months 0, 1, 6 and 12. In patients with more than one TCA available, data were analyzed to compare the changes in ImmuKnow results at the following time points: (i) Months 0 1, (ii) Months 1 6 and (iii) Months 6 12. The effect of rabbit anti-thymocyte globulin (ratg) induction therapy on TCA values over time was also examined. Patients were routinely screened for BKV via urine and blood polymerase chain reaction (PCR) at 1, 6 and 12 months post-transplant or in the event of a clinical suspicion such as an acute rise in serum creatinine. BKV infection was defined as either (i) a positive blood PCR, (ii) a urine PCR viral load >100 million copies/ml or (iii) renal transplant biopsy with interstitial nephritis and viral inclusion bodies that stained positive for SV40 by immunohistochemical staining [16, 17]. This study was performed with the review and approval of the Colorado Institutional Review Board. Statistical analysis TCA values were compared at baseline, 1, 6 and 12 months using t-tests (paired t-tests for changes over time and two-sample t-tests for comparing means at each time point). Categories of TCA (low versus the rest) were compared at 1, 6 and 12 months for those with BKV at time of testing versus samples from patients with no BKV infection at that time using chisquared tests or Fisher s exact test, as appropriate. Results There were a total of 897 ImmuKnow T-cell ATP-release assay (TCA) values available for analysis in 414 renal transplant patients between May 2005 and July 2009 (Table 1). A total of 302 patients (73%) received induction therapy at the time of transplantation, 80% of whom were treated with ratg. At 1-month post-transplant, the majority of patients were on a calcineurin inhibitor-based regimen. Figure 1 demonstrates the range of TCA values over the first year post-transplant. Most values (74%) were between 200 and 599, ng/ml, but values as low as 9 ng/ml and as high as 1104 ng/ml were recorded. Only 23 TCA values were <100 ng/ml. Of 897 values, 122 were drawn Table 1. Baseline characteristics of the 414 patients included in the study Variable Age at time of transplant (year; mean 6 SD) 48.0 6 13.3 Gender Male 253 (61.1%) Female 161 (38.9%) Race African-American 33 (8.0%) Asian 7 (1.7%) Caucasian 297 (71.7%) Hispanic 61 (14.7%) Other 10 (2.4%) Unknown 6 (1.5%) Type of transplant Deceased donor 218 (52.7%) Living related 108 (26.1%) Living unrelated 85 (20.5%) Unknown 3 (0.7%) Induction therapy Yes 302 (72.9%) No 107 (25.9%) a Unknown 5 (1.2%) Type of induction therapy Thymoglobulin 243 (80.5%) Basiliximab 49 (16.2%) Other b 10 (3.3%) Immunosuppression regimen at 1 month c Tacrolimus and mycophenolic acid 298 (72.0%) Tacrolimus and sirolimus 69 (16.6%) Cyclosporine and mycophenolic acid 7 (1.7%) Cyclosporine and sirolimus 2 (0.5%) Other d 34 (8.2%) Unknown 4 (1.0%) a Twelve patients received thymoglobulin within the first month post-transplant for delayed graft function or acute rejection, leaving 95 patients (22.9%) without induction therapy or depleting antibody exposure. b Other induction therapy included OKT3, alemtuzumab, daclizumab or alefacept. c All patients were maintained on prednisone from time of transplant. d Included belatacept or everolimus in addition to CNI or anti-proliferative agent.

Immune function assay trends post-transplant 2567 pre-transplant (baseline), 316 at 1 month, 258 at 6 months and 201 at 12 months post-transplant (Figure 2a). There were 81 patients with TCA values at both baseline and 1 month, 204 patients with values at 1 and 6 months and 157 Fig. 1. ImmuKnow TCA values (n ¼ 897) in 414 renal transplant recipients obtained during the first year post-transplant (CD4 T-cell ATP release, ng/ml). patients with values at 6 and 12 months (Figure 2b). The mean TCA values at each time point are shown in Table 2. When analyzing changes over time in individuals with consecutive measures, mean TCA values increased negligibly from 419 ng/ml at baseline to 441 ng/ml at 1 month (P ¼ 0.32). There was a significant decrease in the mean values between 1 and 6 months post-transplant (466 versus 356 ng/ml, P < 0.001). There was minimal change between mean values at 6 and 12 months post-transplant (357 versus 370 ng/ml, P ¼ 0.33, Figure 2b, Table 2). At each protocol-specified time point, the mean TCA value in patients with newly identified BKV infection (defined as viremia or high-grade viruria) versus those without infection were compared. Additionally, the association of a low (225 ng/ml) TCA result with BKV infection at each time point was determined. The rate of newly diagnosed BKV infection was 15/316 (5%), 20/ 243 (8%) and 10/174 (6%) at 1, 6 and 12 months posttransplant, (removing those who were identified with BKV1 from subsequent assessments). Mean TCA values were similar for BKV1 versus BKV patients at 1 month (mean 406 versus 460 ng/ml, P ¼ 0.25) and 6 months (mean 383 versus 358 ng/ml, P ¼ 0.56) but were significantly lower at 12 months (264 versus 369 ng/ml, Fig. 2. (A) Mean(6standard error) TCA values at baseline, 1, 6 and 12 months. (B) Mean change in TCA value in patients with two consecutive tests. Table 2. Absolute mean and changes in mean TCA values at pre-specified time points 0, 1, 6 and 12 months post-transplant, categorized by BKV status Time of test Mean (SD) TCA value (ng/ml), all patients Mean (SD) TCA value (ng/ml), BKV1 Mean (SD) TCA value (ng/ml), BKV (excluding prior BKV1 patients) P-value, comparing TCA of BKV1 versus BKV Absolute mean TCA values Baseline 391 (186), N ¼ 122 None 391 (186) N ¼ 122 N/A Month 1 458 (182), N ¼ 316 406 (156) N ¼ 15 460 (182) N ¼ 301 0.25 Month 6 355 (174), N ¼ 258 383 (132) N ¼ 20 358 (178) N ¼ 223 0.56 Month 12 363 (160), N ¼ 201 264 (180) N ¼ 10 369 (154) N ¼ 164 0.039 Changes in mean TCA values (P-value for change over time) Baseline to Month 1 419 441 (N ¼ 81, P ¼ 0.32) 395 273 (N ¼ 4, P ¼ 0.26) 421 450 (N ¼ 77, P ¼ 0.19) 0.13 Month 1 to Month 6 466 356 (N ¼ 204, P < 0.001) 511 385 (N ¼ 17, P ¼ 0.02) 464 360 (N ¼ 175, P < 0.001) 0.65 Month 6 to Month 12 357 370 (N ¼ 157, P ¼ 0.33) 314 308 (N ¼ 5, P ¼ 0.93) 367 373 (N ¼ 129, P ¼ 0.66) 0.87

2568 J. Gralla et al. Fig. 3. (A) Mean (6standard error) TCA by BKV status at 1, 6 and 12 months. (B) Change in TCA values in 26 patients with BKV comparing test prior to BKV diagnosis to test at time of BKV diagnosis. Table 3. BKV diagnosed at time of protocol test and corresponding TCA value Time of test and TCA cutoff Number of patients with TCA values a P ¼ 0.039) (Table 2 and Figure 3a). There were a total of 29/316 (9%), 60/258 (23%) and 42/201 (21%) TCA values classified as low (225 ng/ml per manufacturer s definition) at 1, 6 and 12 months post-transplant, respectively. Using the threshold of low TCA of 225 ng/ml, no association was found with BKV at 1 or 6 months, but at 12 months, 17% (6/35) of patients with low values were positive for new-onset BKV compared with 3% (4/139) of patients with TCA values >225 ng/ml (sensitivity 60%, specificity 82%, positive predictive value 17%, negative predictive value 97%, P ¼ 0.005, Table 3). When the definition of low TCA of 150 and 100 ng/ml was examined to test for the association between TCA level and new-onset BKV, none of the 20 patients newly BKV1 at 6 months had a TCA level <150 ng/ml and only 2 of the 10 patients newly BKV1 at 12 months had TCA level <150 ng/ml, limiting statistical interpretation of these lower thresholds of TCA values. When examining trends in TCA over time and the relationship to BKV infection, reductions in TCA of 100, 150 or 200 ng/ml from 0 to 1 month, 1 to 6 months or 6 to 12 months were not associated with BKV infection. At 1 month, only 4 of the 15 newly identified BKV1 patients had both a baseline and a 1-month test for comparison, limiting interpretation at this time point. In one, TCA increased from 258 to 276 ng/ml, while the remaining three decreased from 508 to 132, from 411 to 389 and from 403 Patients with BKV diagnosed at this test Patients BKV at this test (excluding prior BKV1 patients) 1-month TCA 225 ng/ml 29 1 (3%) 28 (97%) 0.99 1-month TCA >225 ng/ml 287 14 (5%) 273 (95%) 6-month TCA 225 ng/ml 55 2 (4%) 53 (96%) 0.26 6-month TCA >225 ng/ml 188 18 (10%) 170 (90%) 12-month TCA 225 ng/ml 35 6 (17%) 29 (83%) 0.005 12-month TCA >225 ng/ml 139 4 (3%) 135 (97%) a Number of tests for each time point do not match those from Figure 1 due to exclusion of patients with BKV infection diagnosed on prior testing. P-value to 293 ng/ml (i.e. from a sample size of four, one patient diagnosed with BKV at 1 month had decreased by >150 ng/ml between the baseline and the 1-month test). For comparison, in 77 patients without BKV at 1 month and baseline TCA values for comparison, 14 (18.1%) sustained a decrease of >150 ng/ml. At 6 months, 17 of 20 patients diagnosed with BKV had both a 1- and 6-month TCA test. Of these, TCA decreased >150 ng/ml in 7/17 (41.1%) patients. Importantly, decreases in TCA were also very common in patients over the 1- to 6-month period, irrespective of BKV status: in the 175 patients without BKV at 6 months with both a 1- and 6-month TCA test for comparison, 67/175 (38.3%) experienced a decrease in TCA of >150 ng/ml. At 12 months, 5 of 10 patients diagnosed with BKV had a 6- and 12-month test for comparison. One decreased (from 522 to 284 ng/ml) and the remaining four increased over this period. Individual plots of the changes in TCA values over time for patients who developed BKV infection are shown in (Figure 3). Separately, the effect of ratg induction therapy on TCA results was assessed. Compared to 95 patients who did not receive any induction therapy and did not receive depleting antibody therapy within the first month for other indications, those individuals who received ratg-depleting antibody therapy (N ¼ 243) had similar TCA values at baseline and 1-month, but significantly lower mean TCA values at both 6 (325 versus 391 ng/ml, P ¼ 0.01) and 12 months

Immune function assay trends post-transplant 2569 (338 versus 412 ng/ml, P ¼ 0.005). When analyzing changes over time in individuals with repeated tests, the use of ratg did not have a significant effect on TCA from baseline to 1 month (406 versus 391 ng/ml, P ¼ 0.55) or between 6 and 12 months (318 versus 341 ng/ml, P ¼ 0.22). However, there was a significant decrease in TCA values between 1 and 6 months (453 versus 327 ng/ml, P < 0.001). These changes in TCA over time were not significantly different from those observed in patients who did not receive any induction therapy. Proportions of patients who developed new-onset BKV were similar among those who received ratg-depleting antibody therapy versus those with no induction therapy at 1 (5.1 versus 4.1%), 6 (8.2 versus 12.9%) and 12 months (4.2 versus 5.4%), respectively. Discussion To our knowledge, this is the largest study analyzing trends in TCA values over time in kidney transplantation, with screening of asymptomatic patients at 0, 1, 6 and 12 months post-transplant. We found that mean values did not change significantly between 0 and 1 month or from 6 to 12 months but did significantly decline between 1 and 6 months, with nearly 40% of patients sustaining a decline in TCA of >150 ng/ml. We correlated TCA values and changes in TCA over time to the degree of immunosuppression, using newly diagnosed BKV infection as a clinical marker of over-immunosuppression. An association with BKV infection (viremia or high-grade viruria) was not identified by either absolute TCA values or changes in TCA over time in the first 6 months, but at 12 months, BKV infection was more prevalent when the absolute TCA value was <225 ng/ml. As depicted in (Figure 2), most patients with BKV infection did have a decrease in TCA prior to diagnosis, but the change in values was similar to the change noted in the cohort without BKV infection (Table 2). This suggests that the ImmuKnow assay may be less helpful as a screening test in asymptomatic patients in the first 6 months post-transplant (due to a consistent pattern of reductions in TCA results that occur during this time in the majority of patients), but after 6 months may have a role in identifying a cohort of low responders for the purpose of targeted monitoring and/or immunosuppression manipulation. The use of depleting antibody induction therapy with ratg leads to similar TCA trends compared to those not receiving induction agents. While the use of ratg therapy did not lead to significant reductions in T-cell function as assessed by the ImmuKnow assay from 0 to 1 month, ratg induction was associated with significantly lower mean TCA values at 6 and 12 months compared to those patients who did not receive induction or depleting antibody therapy. However, this may be related to more aggressive immunosuppression early in the post-transplant course in patients who receive ratg induction rather than the effect of antibody-depleting therapy alone. Our findings with ratg are consistent with those of Serban et al., who measured serial TCA values in 76 renal transplant recipients who received ratg induction therapy during the first year post-transplant. In their series, TCA results were highest at 1-month post-transplant and decreased consistently until 5 months at which time they began to increase slowly [18]. As we have also noted, this trend in those patients who did not receive antibody-depleting agents suggests that cellular immunity (assessed by ImmuKnow) is lowest at ~6 months after transplantation, a time at which the appearance of OIs is considered to be increased [3, 19]. Our findings provide novel insights into the utility of TCA monitoring in the stable post-transplant setting. Several authors have found that low TCA values are associated with infections (both opportunistic and nonopportunistic) at various times post-transplant [8, 9, 11, 13, 18, 20]. Trends in TCA values over time have also been evaluated in a few small studies in patients with infections and shown to increase with reduction in immunosuppression and/or treatment of the underlying infection [10, 12]. For example, Gautam et al. [10] found that in 12 renal transplant recipients with active viral infections, the mean TCA value increased from a mean of 56.8 58.2 ng/ml at the time of infection to 194 118.9 ng/ml with a reduction in immunosuppression and clearance of the virus. In another study of 17 liver transplant patients with infection, the mean TCA values decreased from 333 156 ng/ml prior to the infection to 118 73 ng/ml (P ¼ 0.0014) at the time of infection and then increased (275 119 ng/ml; P ¼ 0.0325) with lower immunosuppression [12]. These studies demonstrate that TCA values are low in the setting of active, clinically apparent infection, and then increase with clinical improvement in disease, but unlike our study do not address use of the TCA in the stable asymptomatic patient for surveillance purposes. In another study of 296 TCA tests in 170 patients during the first 6 months post-kidney transplant, Batal et al. [8] found that in 33 renal transplant recipients with BKV infection (8 with viremia, 25 with viruria), those with BK viremia had significantly lower TCA values than those with viruria or no BKV (102.9 58.6 versus 227.2 146.4 and 231.8 150.8, respectively; P ¼ 0.002). Our results are similar in that our patients first diagnosed with BKV via viremia (N ¼ 31) had lower TCA values than those with high-grade viruria (N ¼ 14) (means 333.0 145.6 versus 432.0 167.9, respectively, P ¼ 0.05) but were higher on average than Batal s cohort (in whom 93% of viremic samples had an associated TCA of <225 ng/ml). Our results indicate that BKV infection can occur despite higher TCA values, particularly in the first 6 months posttransplant. It has been suggested that trends in TCA rather than absolute values of TCA are more predictive of changes in a patient s risk for infection. Longitudinal monitoring of TCA in 50 heart transplant recipients revealed that median values of TCA during periods of rejection (619 ng/ml) and infection (129 ng/ml) were significantly different from TCA values during clinical quiescence (351 ng/ml, P < 0.05). The authors concluded that serial TCA monitoring rather than single point estimates were more useful in interpreting an individual s immune status [20]. We did not identify a trend in TCA over time in those with versus without BKV infection. Unfortunately, in the prior study, the testing protocol was not specified, and trends over time for individuals with adverse events were not compared to a

2570 J. Gralla et al. cohort without infection or rejection, raising the question of the natural history of TCA responses over time in heart transplant recipients and whether those with clinical events were significantly different in their TCA changes from those without clinical events. We acknowledge that our study has a number of limitations. TCA values and BKV assessment were checked pretransplant and then at 1, 6 and 12 months but were not universally collected in every patient at every time point. Due to the cost of BKV testing and the evolving coverage by health insurers in the USA during the period of this study as well as the extended geographic distance of our patient population to our center, more frequent testing was not feasible. Screening more frequently might identify individuals with rapidly changing immune responsiveness who may be at heightened risk for complications, such as intercurrent BKV infection at the 3- to 6-month time frame. While we did not find an association of absolute TCA values <150 or decreases in TCA of 100, 150 and 200 ng/ml to be predictive of BKV infection, very few patients had absolute TCA values of <100 or decreases of >200 ng/ml over time, so we cannot exclude that greater decreases in TCA could be more predictive of infectious complications. We only examined BKV infection due to the objective data collection and well-accepted nature of BKV infection, its progression from viruria to viremia to clinical disease and its association with over-immunosuppression [16, 21] but did not include other infections for this analysis. It is possible that other infections such as CMV or EBV behave in a different manner with respect to TCA dynamics. Since our center does not perform protocol-based PCR screening for these OIs in the asymptomatic setting, we cannot comment on the potential utility of TCA monitoring for risk of these infections. In summary, this large single-center experience with Immuknow assay monitoring in stable renal transplant patients during the first year post-transplant shows significant reductions in assay values from 1 to 6 months post-transplant in many individuals that render interpretation of changes in Immuknow results difficult when attempting to identify patients at risk for over-immunosuppression during this period. Persistently, low values after 6 months may be suggestive of an over-immunosuppressed state associated with an increased risk of OIs, such as BKV. Based on these results, targeted monitoring of patients with low values following planned immunosuppression reduction (e.g. 6 months) may be a reasonable next step in determining the utility of ImmuKnow monitoring in the asymptomatic patient. Acknowledgements. Funding. No external funding was provided for the research or creation of this manuscript. J.G. was supported by The Children s Hospital Research Institute. 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Transplantation 2005; 79: 1277 1286 Received for publication: 1.4.11; Accepted in revised form: 25.10.11