Introduction
Following hematopoietic stem cell (HSCT) and solid organ transplantation (SOT), immunosuppressive therapy is administered to prevent graft rejection and graft-versus-host disease (GvHD). Prophylactic regimens transiently lead to strong immunosuppression, mainly, by decreasing CD3
+ T-cell numbers [
1]. Consequently, the risk of life-threatening bacterial, fungal, and viral infections as well as recurrent viral reactivation increases. Additionally, lymphopenia in the regeneration phase after HSCT enhances the pathogen-associated morbidity and mortality. Up to 22% and 53% of overall mortality after HSCT and SOT, respectively, are associated with infections resulting from a lack of specific T-cell immunity [
2‐
4]. Individuals with congenital primary or secondary immunodeficiencies are even more susceptible to infectious complications, which are among the leading causes of death [
5‐
7].
The main viral pathogens causing infection-related deaths in patients with immunodeficiency or after transplantation are endogenous herpesviruses such as cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV6); lytic viruses like adenovirus (ADV); as well as polyomaviruses such as the BK (BKV) and JC virus (JCV) [
7‐
14]. CMV reactivation observed in 40–65% of CMV-seropositive recipients after HSCT is associated with a higher risk of mortality [
15‐
17]. Incidence rates of EBV reactivation and post-transplant lymphoproliferative disease (PTLD) vary from 0.1 to 63%, depending on the type of transplant [
17‐
20]. In patients with immunodeficiencies, especially severe combined immunodeficiencies, fatality rates from severe and recurrent pulmonary ADV infections as well as disseminated disease have been reported to be up to 55% [
1,
21]. High-level HHV6 reactivation after allogeneic HSCT has been described in 30–50% of recipients [
22,
23]. BKV and JCV viremia occur in 54% and 25% of HSCT recipients, respectively, JCV viruria in 3.8–40% of kidney transplant patients, and BKV-induced nephropathy in 5.3% of the patients [
17,
24,
25]. JCV reactivation in transplant and non-transplant patients can result in life-threatening progressive multifocal leukoencephalopathy (PML), with mortality rates of up to 71% [
26‐
28]. Other herpesviruses like herpes simplex virus type 1 (HSV1), herpes simplex virus type 2 (HSV2), and varicella-zoster virus (VZV), along with respiratory RNA viruses such as influenza A virus (IAV) and human respiratory syncytial virus (RSV), expose immunocompromised individuals to a constant risk of severe and potentially life-threatening complications [
29‐
32].
Over the last decades, advances in antiviral drug therapy and prophylactic and pre-emptive antiviral treatment strategies have decreased infectious complications in immunocompromised patients. However, they are associated with toxic side effects and ineffective in case of drug resistances. Moreover, due to insufficient reconstitution of cellular immunity, viral infections can only be controlled but not completely eliminated [
16,
33,
34]. A major clinical challenge remains the complex interplay between immunosuppressive treatment and the maintenance or establishment of antiviral immunity [
8]. Therefore, clinicians must carefully balance the risks of graft rejection or GvHD on the one side and the maintenance of protective immunity on the other.
Currently, information about virus-specific T-cell (VST) frequencies required for virus control and clearance is scarce and empirical data on protective pathogen-specific T-cell numbers in blood are highly desirable. Individualized antiviral treatment strategies require knowledge about VST frequencies since it helps clinicians assess the effects of modalities such as antiviral drug therapy, and weigh the opportunity or need for reduction of immunosuppression or adoptive T-cell transfer (AT) [
35‐
37]. If the VST frequencies of an immunosuppressed patient are within normal ranges of healthy donors, antiviral drug therapy presents a successful strategy. With VST frequencies below average, cellular therapies, such as AT, offer a promising approach. Reference ranges will help clinicians to predict responses to AT [
2,
38,
39].
The aim of this study was to provide data on VST frequencies in a population of healthy donors (
n = 151) as an aid to therapeutic decision-making in immunocompromised patients and patients with immune disorders. Antiviral T cells against 11 clinically highly relevant viruses were determined by interferon-gamma (IFN-γ) enzyme-linked immunospot (ELISpot) and characterized regarding frequency, phenotype, age, and gender. Moreover, ELISpot data were correlated to serological testing routinely performed for 7 of the 11 viruses. All donors were seropositive for at least four viruses, and the spectrum of antiviral immunity increased with age. Overall, VST frequencies were higher for DNA and persistent viruses (CMV, EBV), lower for RNA viruses (RSV, IAV), and lowest for BKV and JCV. The reference values established in this study give clinicians a valuable tool for interpreting a patient’s specific antiviral T-cell profile, and for estimating the need for and type of further therapeutic interventions, which could potentially be a breakthrough in the evaluation of immune status [
40‐
43].
Discussion
Immunocompromised HSCT and SOT recipients and individuals with congenital primary or secondary immunodeficiencies present a high burden of mortality due to life-threatening bacterial, fungal, and viral infections as well as recurrent viral reactivations [
1,
5,
7]. Although antiviral drug treatments have advanced over the years, they are still associated with toxic side effects. In the absence of functional VSTs, they can only control but not completely eliminate viruses [
16,
33,
34]. Antiviral T-cell frequencies determine whether treatment (e.g., antiviral drug therapy, reduction of immunosuppression, or AT) is needed [
35‐
37]. This study aimed to establish reference values for antiviral T-cell frequencies against 11 clinically relevant human viruses in healthy donors. These data should help to improve the prevention and treatment of viral complications, leading to better outcomes in HSCT and SOT recipients and patients with immune disorders.
In the transplant setting, determination of infectious disease markers for viruses mainly includes the CMV and EBV serostatus of donor and recipient. Donor seropositivity provides an opportunity for transfer of antigen-specific T cells to improve immunity in HSCT recipients with insufficient endogenous antiviral immunity. Conversely, SOT from seropositive donors to negative recipients is associated with an increased risk of primary viral infections in immunosuppressed SOT recipients. In this study, the discrepancies between ELISA-based CMV-IgG assay and immunoblotting as well as ELISpot assay for CMV indicated a false-positive rate of 13.1% for the CMV-IgG assay. As confirmed by other studies, standard serology tests might not be reliable enough for adoptive immunotherapy when no peptide-specific memory T cells are present [
44,
46]. Contrary to other studies [
44,
47], we did not detect any CMV-specific T cells in seronegative donors, but the lack of detection might be due to the relatively short restimulation. Moreover, some donors with VSTs against a given virus were categorized as seronegative by ELISA, particularly for ADV, HSV, VZV, RSV, and IAV, which might be due to utilization of different, less concentrated surface antigens in ELISA compared to ELISpot. For instance, here, the utilized ADV-IgG assay covers ADV type 2 while ADV5_Hexon and ADV5_Penton peptide pools are derived from ADV type 5. Conversely, it might explain the lack of functional VSTs in some donors identified as ADV-seropositive. Similar circumstances presented for HSV, VZV, RSV, and IAV. Overall, majority of seropositive donors had the respective VSTs and their frequencies were significantly higher compared to seronegative donors. Furthermore, T-cell and antibody production is dependent on reinfection and recall immunization, which might not have occurred in these donors. However, one study using the HSV immunoblot assay suggests that exposure to HSV can induce HSV-specific cellular immunity without seroconversion [
48]. This may also explain the observed discrepancy between HSV serostatus and the detection of HSV-specific T cells.
High frequencies of herpesvirus-specific T cells were detected in the present study, suggesting that viruses causing persistent infections are able to generate higher T-cell frequencies. Others have observed comparably high frequencies of VSTs for CMV [
49] and EBV in healthy seropositive donors [
50]. The ability of these viruses to achieve latency and frequently initiate productive replication cycles was shown in murine models [
51]. This mechanism provides a continuous stimulus for the maintenance of VSTs. The positive correlation within the group of herpesviruses found in this study has been demonstrated previously [
52‐
55]. It has been hypothesized that their high co-prevalence is associated with increased age or lower socioeconomic status [
52]. Our study confirmed the association between age and seroprevalence, but did not assess socioeconomic factors. The majority of EBV-seropositive donors in the present study had EBV_Consensus-specific T cells. In contrast to the EBV-derived overlapping peptide pools covering the entire sequence of the respective protein, Consensus contains a mix of peptides derived from 13 lytic and latent EBV proteins, and covers 14 frequent HLA class I and II molecules. The use of overlapping peptide pools allows for detection of T-cell responses to multiple epitopes regardless of HLA type [
56,
57], while the use of an antigen pool derived from various proteins with different HLA restrictions leads to a greater T-cell response with a higher range of clinically relevant VSTs due to its high antigenic diversity [
58]. EBNA1-specific T cells are of utmost clinical importance since EBNA-1 plays numerous roles in EBV latency and is the only EBV protein expressed in all EBV-associated tumors [
59,
60]. JC polyomavirus, which establishes persistent infections in the kidney and lymphoid organs, normally remains dormant but can reactivate in immunocompromised individuals where it can cause PML, a life-threatening infection of the brain [
26‐
28,
61]. In this study, JCV yielded the lowest VST frequencies of all viruses tested. JCV-specific VST frequencies were reported to be low, but without sufficient evidence [
62,
63]. For IAV, a virus not typically causing long-term latent or persistent infections, we observed higher frequencies of antigen-specific T cells than for BKV and JCV. This finding contradicts the notion of a correlation between general viral latency and higher immune response. Herpesviruses are among the few viruses capable of true latency, i.e., persistence and reversibility, characterized by reactivation of expression of the entire viral genome under certain conditions [
64]. The results of the present study suggest that true viral latency is associated with the generation of higher VST frequencies.
In this study, IAV and RSV were characterized by low VST frequencies. Both are RNA viruses infecting cells by directly releasing RNA into the cytoplasm of host cells [
65]. Once inside, viral proteins can be replicated without transcribing viral DNA into RNA, unlike DNA viruses. Many RNA viruses do not elicit long-lasting immune protection after infection due to their innate immune evasion strategies and can cause a reoccurrence of symptoms. While IAV is able to elicit protective immunity, its genetic drift and shift usually lead to inadequate immune responses after reinfection. Because the affected immune response also impacts subsequent adaptive responses, viral innate immune evasion often undermines fully protective immunity [
66].
Even though BKV belongs to the group of viruses with generally low VST frequencies [
67], it is a major complication after kidney transplantation and therefore of high clinical relevance [
17,
24,
68]. Despite high sequence homology between BKV and JCV, VST frequencies for JCV were lower compared to BKV. In line with previous studies, we observed a correlation between BKV- and JCV-reactive T cells [
69,
70], implicating high potential of BKV-specific T cells for treatment of both BKV- and JCV-associated diseases such as PML, where third-party BKV-specific T-cell transfer has shown promising results [
28,
71,
72].
The phenotypic structure of VSTs involved in different viral infections and reactivations varies due to differences in response patterns. In our study, T
N frequencies were generally higher in individuals lacking antiviral T cells and in seronegative donors. It has been reported that while EBV- and HSV-specific T cells included higher ratios of CD8
+ T cells, CD4
+ T cells were the dominant T-cell subset in ADV, BKV, and VZV [
73‐
77]. The importance of T-cell subsets varies depending on the virus and the associated disease and needs to be considered when evaluating patient immune status.
Our findings confirmed that antiviral immunity increases with age and that seroprevalence is higher among older individuals [
78]. While age is associated with a highly differentiated T-cell repertoire because the cumulative number of contacts to viral agents increases over time [
79], the process of immunosenescence leads to an age-related decrease in immune system activity, including T-cell function, despite higher effector T-cell frequencies [
78,
80,
81]. Consequently, the decline in immune function is believed to increase the risk of viral infections and reactivations, leading to higher mortality rates among the elderly [
40,
82]. Here, we determined the frequencies of IFN-γ-producing, functional, antiviral T cells, thereby—at least in part—accounting for the possible loss of immune function associated with aging. However, our cohort did not cover the entire age range. Characterization of the antiviral T-cell repertoire of older individuals requires the inclusion of additional factors like T-cell senescence, exhaustion, and additional effector molecules. However, as corroborated by other studies, our results indicate a correlation between age and VST frequency, possibly caused by more frequent and/or recurrent viral infections over time [
53,
83,
84]. In contrast, younger individuals with no history of exposure to a broad variety of antigens often suffer from severe viral infections after transplantation due to the lack of endogenous antiviral T cells. In particular, EBV causes PTLD in many young patients after transplantation [
20,
85]. Due to their limited antiviral T-cell repertoire, further studies are needed to determine the VST frequencies for adjusting antiviral or immunosuppressive treatment strategies in young patients.
This study aimed to improve the clinical applicability of antiviral T-cell frequencies by characterizing T cells specific to clinically relevant viruses in terms of numbers as well as age and gender distribution in a great cohort of healthy donors. In line with previous studies, this study demonstrated that antiviral immunity increases with age. Furthermore, a positive correlation within herpesviruses was found. With exception of CMV_pp65, positive serology was not necessarily equivalent to detection of the respective VSTs. The findings of this study have important implications for the evaluation of T-cell mediated immunity and treatment decision-making to determine the need for antiviral treatment or reduction of immunosuppression. Together, this data will improve the outcome of immunocompromised patients and provide better comparability of currently used immunogenic stimulants regarding clinical outcome.
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