Validation of T-Track® CMV to assess the functionality of cytomegalovirus-reactive cell-mediated immunity in hemodialysis patients

Cytomegalovirus (CMV) is a major cause of infectious complications in immunocompromised individuals. Protection against CMV infection or reactivation is normally assured by both the innate and adaptive arms of the immune system [1, 2]. While the humoral and innate responses are essential for the early response to infection [1, 3, 4], cellular immunity is required to control latency and prevent CMV reactivation in latently infected individuals [1]. CD8⁺ cytotoxic T cells (CTL) and CD4⁺ T helper (Th) cells are both required to assure efficient immune protection against CMV reactivation [1, 5‐8]. Primary infection is dominated by CD8⁺ T cell response, preferentially targeting CMV immediate early-1 (IE-1) antigen, while long-term recovery is dominated by CD4⁺ T cell response and a switch of reactivity toward CMV lower matrix phosphoprotein 65 (pp65) [6, 8‐11]. The frequency of CMV-specific CD8⁺ and CD4⁺ T cells is highly variable, both between healthy CMV-seropositive individuals and during the course of CMV reactivation, and correlates with varying levels of protection [6, 9, 11‐13]. Beside changes in T cell frequency, alterations in T cell functionality are associated with impaired response to chronic viral infection [14‐17].

Functional impairment of cell-mediated immunity (CMI) in the course of immunosuppression, such as in solid-organ transplant recipients, is a major cause of uncontrolled CMV replication and related clinical complications [18‐21]. Treatment regimens with antivirals are costly and associated with harmful side effects. Assessment of CMV-specific immunity may be beneficial to identify patients at increased risk of viral complications, possibly allowing personalized adjustment of antiviral and immunosuppressive therapies.

Various experimental approaches exist to measure CMV-specific CMI. Direct T cell staining with for instance class I iTAg™ MHC Tetramers (Beckman Coulter) allows the quantification of epitope-specific CD8⁺ cells by flow cytometry [18, 22‐24]. The sensitivity of tetramer-based assays strictly depends on the coverage of the patient population by the selected HLA types, and this method cannot assess the functionality of CD8⁺ cells. Several assays assessing CMV-specific T cell function have been described. Principally, they measure the production of activation markers (e.g. cytokines such as IFN-γ) in response to antigen stimulation, using intracellular cytokine staining followed by flow cytometry [6, 7, 12, 13, 25, 26], ELISA [21, 27‐30], or ELISpot [31‐33] assays. These approaches differ not only in their read-out format but also in the antigen (peptide vs. protein) used for the ex vivo stimulation. Peptide-based immune monitoring tests such as QuantiFERON®-CMV (Qiagen) allow the quantification of IFN-γ produced by epitope-specific CD8⁺ T cells. Whole blood samples are stimulated with a pool of 22 immunogenic peptides (mapping at IE-1, IE-2, pp28, pp50, pp65 and gB CMV antigens) and covering > 98% of HLA class-I haplotypes. Reactive CD8⁺ T cells are monitored by quantifying secreted IFN-γ by ELISA [34]. QuantiFERON®-CMV was used in a number of studies to assess the risk of CMV reactivation and related disease following solid-organ transplantation [21, 27‐30]. A disadvantage of QuantiFERON®-CMV is that it does not assess CMV-specific CD4⁺ T cell function and that it often yields indeterminate results that cannot be interpreted [28, 35, 36]. T-Track® CMV is based on the stimulation of freshly isolated peripheral blood mononuclear cells (PBMC) with recombinant urea-formulated (T-activated®) immunodominant CMV IE-1 and pp65 proteins, and the subsequent quantification of antigen-reactive effector cells using an IFN-γ ELISpot assay. T-activated® proteins (aproteins) are processed via the exogenous and endogenous antigen processing pathways, resulting in the presentation of naturally-generated peptides by MHC class I and class II molecules, thus enabling the stimulation of a broad spectrum of CMV-protective cells including CD8⁺ and CD4⁺ T cells, as well as the bystander activation of NK and NKT-like cells [37, 38]. As such, T-Track® CMV is not restricted to particular HLA types. Performance of T-Track® CMV has been recently characterized [38]. A recent study demonstrated its high sensitivity in evaluating changes in CMV-specific CMI during and after pregnancy [39].

Primary aim of this cross-sectional prospective multicenter study was to evaluate the suitability of T-Track® CMV to assess the functionality of CMV-specific CMI in a cohort of patients on hemodialysis due to end-stage renal failure, and thus being representative of patients prior to renal transplantation. Secondary aim of the study was to compare the performance of T-Track® CMV to that of QuantiFERON®-CMV and iTAg™ MHC Tetramers in terms of positive and negative agreement with CMV serology (gold standard reference).

T-Track® CMV represents a novel assay format, which relies on the functional assessment of various CMV protein-reactive effector cells, including CD4⁺ (Th) cells, CD8⁺ (CTL), NK and NKT-like cells [37, 38], all of which being described to contribute to the clearance of CMV replication [1, 6‐8, 41‐44]. In this study, the suitability of T-Track® CMV to measure CMV-specific CMI in a cohort of dialysis patients and its performance against QuantiFERON®-CMV [34] and iTAg™ MHC Tetramer assays [24] were evaluated.

T-Track® CMV revealed a positive agreement with CMV-serology of 90% in hemodialysis patients, higher than that measured with QuantiFERON®-CMV (73%) and a mixture of 6 preselected CMV-specific iTAg™ MHC Tetramers (77%), indicating a higher sensitivity of T-Track® CMV compared to QuantiFERON®-CMV and iTAg™ MHC Tetramers. This difference in positive agreement with CMV-serology is likely due to the difference in format of the three assays. Beside the detection of a broad repertoire of CD8⁺ T cells as a result of antigen cross-presentation [37], T-Track® CMV is indeed able to assess the functionality of CMV-reactive CD4⁺ cells but also the bystander activation of IFN-γ-producing NK and NKT-like cells [41, 43‐46]. In contrast, QuantiFERON®-CMV and iTAg™ MHC Tetramers are restricted to the detection of selected CMV-specific CD8⁺ cells. In addition to the assay format, the combination of results of the separate measurement of pp65- and IE-1-responsive effector cells by T-Track® CMV contributes to the increased positive agreement with CMV serology, from 87% with pp65-specific CMI alone to 90% with both pp65- and IE-1-specific CMI. This positive contribution of IE-1 to the sensitivity of T-Track® CMV is in agreement with the demonstration that CMV-seropositive healthy donors do not always exhibit a pp65-specific CD8⁺ T cell response and that a non-negligible proportion of individuals only show a CD8⁺ T cell response to IE-1 [47]. Other factors potentially enhancing the sensitivity of T-Track® CMV are the standardization of the assay, which uses a constant number of PBMC (as opposed to whole blood in QuantiFERON®-CMV, possibly resulting in high inter-individual variability), its HLA-type-independence (as opposed especially to the iTAg™ MHC Tetramer assay) and the absence of indeterminate results (as opposed to QuantiFERON®-CMV). In that regard, 4/66 CMV-seropositive and 2/57 CMV-seronegative hemodialysis patients yielded indeterminate results with QuantiFERON®-CMV, which - with a rate of 5% - is lower than what was reported in transplant recipients [28, 35, 36].

Interestingly, the positive agreement of T-Track® CMV with CMV-serology of 90% measured in this cohort of hemodialysis patients was lower than that obtained in CMV-seropositive healthy individuals (97%; [38]). Similarly, the positive agreement of QuantiFERON®-CMV results with CMV-serology in hemodialysis patients (73%) is below the positive agreement of 88% to 97% previously reported in healthy adults [27, 34]. This difference might be explained by a functional impairment of Th cells, CTL, Antigen-presenting cells (APC), NK and NKT cells in patients with end-stage renal failure undergoing hemodialysis [48‐52]. A reduced CMV-CMI prior to renal transplantation might be associated with an increased risk of CMV reactivation following transplantation. In support to this proposition, several studies reported an association between impaired CMV-specific CMI pre-transplantation and increased risk for CMV viremia post-transplantation [29, 31, 53]. The high positive agreement of T-Track® CMV with CMV serology observed in this cohort of hemodialysis patients therefore emphasizes the suitability and clinical relevance of T-Track® CMV for patients eligible for renal transplantation.

Although both CMV pp65 and IE-1 antigens contain multiple CD4⁺ and CD8⁺ T cell epitopes presented by different HLA alleles [40], the number of reactive effector cells responding to stimulation with app65 was substantially higher than that responding to aIE-1. This difference might result in part from the dynamics of pp65- and IE-1-reactive CD4⁺ and CD8⁺ T cells in the course of the immune response to CMV infection, long-term seroconversion being dominated by pp65- over IE-1-reactive T cells [8‐11, 53, 54]. Moreover, mechanisms of immune evasion involving CMV-encoded unique short (US) proteins and resulting in the inhibition of the MHC-I-dependent antigen presentation pathway appear to be responsible for impaired IE-1 antigen processing and presentation, and thus in the low frequency of IE-1-reactive CD8⁺ T cells [55‐57]. On the other hand, differential antigen uptake, processing and presentation by APC, possibly influenced by pp65 and IE-1 intrinsic properties [54, 58, 59], might explain inter-individual differences in the frequency of CMV antigen-specific T cells. Accordingly, comparable CD8⁺ T cell response to IE-1 and pp65 has also been described in some CMV-seropositive healthy donors [47, 60]. The clinical significance of the differential responses to different antigens using T-Track® CMV needs to be elucidated in future studies.

12/57 CMV-seronegative patients revealed positive T-Track® CMV results, corresponding to a negative agreement of T-Track® CMV with CMV serology of 79%. Positive test results were mainly attributed to aIE-1 stimulation, and negative agreement raised to 98% when considering the results of app65 stimulation alone. IE-1-induced spot counts were close to T-Track® CMV positivity threshold in 9/12 patients and only 3 CMV-seronegative patients showed higher IE-1-induced spot counts. We can reasonably rule out false negative CMV serology test results in these patients, as repetition of CMV serology 6 months upon completion of the study in 9 patients who were still available, confirmed their negative serostatus for IgG and IgM (data not shown). Comparatively, 7/55 QuantiFERON®-CMV and 3/45 CMV iTAg™ MHC Tetramer measurements also revealed positive test results in seronegative dialysis patients. Interestingly, with one exception, the 12, 7 and 3 CMV seronegative patients with positive test results in T-Track® CMV, QuantiFERON®-CMV and CMV iTAg™ MHC Tetramers, respectively, were different. This inter-assay variability contributes to the moderate agreement (κ = ~0.4) observed between T-Track® CMV and the 2 alternative assays, and is in agreement with previous studies reporting discordant results between IFN-γ ELISpot and QuantiFERON®-CMV [61, 62]. This inter-assay variability likely reflects differences in the ability of antigen stimulants in each assay to activate distinct subsets of CMV-reactive T cells. Urea formulation of T-activated® CMV antigens increases protein uptake and promotes antigen processing and presentation in the context of both MHC-I (cross-presentation) and MHC-II [37]. T-Track® CMV is thus able to activate a broad range of CD8⁺ and CD4⁺ T cells, encompassing a larger T cell repertoire than QuantiFERON®-CMV or iTAg™ MHC-I Tetramers, and possibly explaining the higher number of CMV-seronegative patients with positive T-Track® CMV test results.

Interestingly, detection of CMV-reactive effector T cells within CMV-seronegative individuals has been described by others, both in healthy individuals and in transplant recipients, at frequencies of 2–11% among healthy donors and up to 30% in renal transplant recipients [13, 63‐65]. With 21% CMV-seronegative hemodialysis patients with positive T-Track® CMV results, our data are in concordance with these published studies. Although Sester et al. questioned the accuracy of serologic testing, in particular in case of borderline immunoglobulin titers [65], Loeth et al. elegantly demonstrated in their study on healthy individuals, that the frequency of 11% seronegative donors with pp65-specific CD4⁺ and CD8⁺ response was neither due to wrong serological assignment, nor to immune cross-reactivity with the closely related herpes virus HHV6, nor to in vitro priming. Instead, their demonstration that a large proportion of seronegative donors could mount a strong pp65-specific CD4⁺ (and to a lesser extent CD8⁺) response upon in vitro stimulation, led the authors to suggest that these individuals were previously exposed to CMV but failed to mount a humoral immune response [63]. We cannot exclude at this point this possibility nor that TCR cross-reactivity with closely related herpes viruses [66, 67] or with environmental antigens [68, 69] is responsible for the detection of CMV-reactive cells in CMV-seronegative hemodialysis patients. On the other hand, we can reasonably exclude the possibility that signals detected by IFN-γ ELISpot following 19 h of antigen stimulation originate from CMV-specific naïve T cells present in the PBMC population [70‐72]. Indeed, although antigen-specific naïve T cells can be primed and expanded in vitro and in vivo [69, 72‐78], the vast majority of antigen-stimulated naïve T cells produce no IFN-γ and do not divide for the first ~3 days of stimulation [69, 76, 79‐82]. The higher proportion of CMV-reactive cells in seronegative dialysis patients in T-Track® CMV compared to QuantiFERON®-CMV and iTAg™ MHC Tetramers supports the hypothesis that IE-1-specific CD4⁺ T cells (and possibly CD8⁺ T cells through cross-presentation) contribute to the detected signals. On the other hand, the increased proportion of IE-1-reactive cells over pp65-reactive cells in CMV-seronegative patients supports the idea of a recent exposure to CMV, as response to primary infection is usually dominated by IE-1-reactive (predominantly CD8⁺) effector cells [6, 8‐11]. Additional experiments will be necessary to address these propositions. Whatever the mechanism involved in the generation of CMV-reactive effector cells in CMV-seronegative patients, these observations raise the attractive possibility that these individuals might have a protective immunity against CMV infection. Clearly, further investigations are needed to address this possibility. Altogether, our data suggest that T-Track® CMV exhibits a performance superior to that of QuantiFERON®-CMV and of iTAg™ MHC Tetramers, and possibly also superior to that of CMV-IgG serology, for the detection of possible immunity against CMV.

T-Track® CMV represents a highly standardized and sensitive assay suitable for the monitoring of CMV-specific cell-mediated immunity in end-stage renal failure patients, representative of patients prior to renal transplantation. Further validation of T-Track® CMV in multi-center clinical studies on kidney and allogeneic stem cell transplant patients is currently on-going, to evaluate its use for the risk assessment and prediction of CMV-related clinical complications in transplant recipients. In these situations, monitoring of CMV-specific CMI could help physicians better define patient populations that would benefit from prophylactic antiviral therapy, and assist the decision as to when to withdraw prophylaxis safely. Reducing prophylactic antiviral treatment would be beneficial in limiting both treatment-related nephrotoxic side effects and costs.