This pilot study represents the first longitudinal evaluation of the kinetics of global and CMV-specific T-cell responses in a cohort of pediatric SOT recipients. Our results indicate that ELI-SPOT assays are feasible to perform in pediatric populations and may be a useful tool to predict risk for CMV disease. CMV DNAemia developed in three of the 10 subjects (30%) studied longitudinally and was associated with loss of global CD3 response in the two who exhibited sustained DNAemia. This observation suggests that IFNG responses to anti-CD3mAB may provide a biomarker to identify those patients at risk for persistent CMV replication and its sequelae. Monitoring CD3 responses may have broader applicability than evaluating pp65 responses as they are independent of CMV serostatus, HLA type, and selection and testing of appropriate peptides for ex vivo stimulation.
This study also provides insights into the kinetics of functional and quantitative T-cell recovery in pediatric transplantation patients. There was a significant depression in ELISPOT responses and Th1 and Th2 cytokine secretion following stimulation of PBMC with anti-CD3mAb one month post-transplant, which recovered by six months. IFNG responses at six months were greater than pretransplant levels, possibly reflecting improvement in overall health and reduction in IS. However, depression in the frequency of CD4+ cells persisted for at least six months post-transplant. Furthermore, the CD4/CD8 ratio remained significantly lower in children who were more than one yr post-transplant when compared with HC. The decline in CD4+ cells in the early post-transplant period likely reflects the susceptibility of rapidly dividing CD4+ cells to Th (44
), whereas the lag in recovery of CD4/CD8 ratio may reflect the impact of maintenance immunosuppressive therapy (46
). These more modest and persistent abnormalities in CMI may contribute to overall impaired memory responses and increased susceptibility to infections.
Recovery of CMV-specific responses is more difficult to evaluate because of greater variability in the IFNG ELISPOT responses to pp65 in CMV-seropositive subjects compared with anti-CD3mAb responses. This variability was also evident in HC and LTTx cohorts () and may reflect differences in the frequency of sub-clinical reactivation leading to variable boosting of CMV-specific T-cell responses among subjects (47
We are unable to discern whether the reduction in CMI responses in the two subjects who developed persistent CMV DNAemia was triggered by viral replication or whether the decrease in CMI responses predisposed these subjects to CMV. However, our findings support the notion that preemptive monitoring of T-cell responses may still be a useful indicator of risk for CMV disease. While no prior studies have been reported in pediatric SOT recipients, a study of 108 adult SOT recipients found IFNG responses to be a significant predictor of CMV disease. During the first three months following transplantation, only 5% of subjects with IFNG responses to ex vivo
stimulation with CMV peptides developed CMV disease, whereas 23% of subjects without IFNG responses developed CMV disease (p = 0.038) (28
Evaluating CMV-specific CD4+ and CD8+ T-cell responses may also identify transplant subjects at risk for other CMV-related morbidities, such as secondary infections or graft dysfunction. In a study of 19 CMV-seropositive adult heart transplant recipients, delayed reconstitution of CMV-specific CD4+ cells was associated not only with higher peak CMV viral loads but also with greater risk of acute rejection and transplant arteriopathy (48
Monitoring both global and CMV-specific T-cell responses could inform and guide clinical care practices during the post-transplant period. The inclusion of global CMI responses may be particularly beneficial for pediatric transplant recipients, who are often seronegative prior to transplantation, and therefore have minimal immunological memory. Universal prophylaxis has a number of associated complications; immune monitoring could identify those subjects who reconstitute their CMI responses early and could therefore discontinue prophylaxis and be followed preemptively. Conversely, subjects with delayed immune reconstitution may benefit from more prolonged prophylaxis and more aggressive monitoring. Furthermore, once a patient manifests CMV DNAemia or tissue-invasive disease, monitoring global and CMV-specific CMI could potentially predict severity of disease and timing of recovery. For example, persistently poor CMV-specific T-cell responses in the cardiac transplant recipient who developed DNAemia and hepatitis () could have predicted the subject’s clinical relapse and the possible need for additional antiviral therapy or more aggressive reduction of IS.
Assessing global T-cell responses could also have implications for management of other OI and timing of vaccinations post-transplant. For example, the duration of prophylaxis for pneumocystis jirovecii (PCP) is controversial and varies by transplant center. Similarly, data are limited regarding the optimal time to safely and effectively vaccinate pediatric SOT recipients. Restoration of T-cell numbers and global function could provide a biomarker to determine duration of prophylaxis of other OI or when vaccinations, particularly live attenuated vaccines, can be safely and effectively administered.
Advances in functional immunoassays for evaluation of CMI have greatly facilitated immunologic studies of immunocompromised pediatric patients. The ELISPOT and multiplex cytokine assays offer the advantage of relatively simple methodology and high reproducibility (49
). Despite the advantages of more detailed analyses of T-cell populations with flow cytometry, intracellular cytokine staining, and tetramer staining, these additional studies often require greater volumes of blood than are typically available from younger pediatric transplant patients. Although functional T-cell assays are technically demanding and expensive, it is anticipated future advances in automation will render CMI testing more feasible. The ELISPOT and multiplex cytokine assays used in this study required only 2–3 mL of blood and provide a relatively rapid approach to evaluate CMI function. Many centers now offer comparable assays for other pathogens as part of their laboratory portfolio, such as the QuantiFERON test for latent TB.
Our study has several additional limitations. First, the number of children in the longitudinal cohort was small. Secondly, although pp65 is considered an immunodominant epitope and use of pp65 overlapping peptide pools has been shown to increase the sensitivity and specificity of detecting CMV-specific immune responses, not all subjects may have recognized the represented epitopes (51
). Evaluating responses to other CMV antigens, including the immediate early antigens, may further characterize these responses. Finally, follow-up periods beyond six months would clarify the dynamics of reconstitution of T-cell responses during the late transplant period. Larger prospective studies are necessary to fully evaluate the quantitative and qualitative T-cell responses required for control of viral replication and to determine the level of global or virus-specific T-cell responses necessary to prevent CMV and its complications.