To the Editor:

A number of factors interact to determine the risk of viral infections/reactivations following allogeneic hematopoietic stem cell transplant (HSCT). In the last decade, this issue became even more relevant with the introduction of new transplant platforms, including in particular the growing use of Post-Transplant Cyclophosphamide (PT-Cy) as graft-versus-host disease (GvHD) prophylaxis [1]. Although the recovery is fast and robust among patients treated with PT-Cy, this approach may affect the dynamics and final output of immune reconstitution, as well as impact the risk of infections, exposing HSCT recipients to higher incidence of viral infections [2, 3].

Cytomegalovirus (CMV) represents a relevant complication in HSCT recipients with PT-Cy [4], although the recent advent of letermovir prophylaxis has profoundly changed CMV presentation also in this context [5,6,7,8,9]. Although rare, human herpesvirus 6 (HHV-6) [10] and adenovirus (AdV) [11] have emerged as additional threats in HSCT recipients. This changing epidemiology of viral infections poses new challenges, from post-transplant monitoring to potential prophylactic/therapeutic approaches.

In this context, post-transplant immune reconstitution plays a major role in determining the outcome of HSCT recipients [12]. Immune cell counts provide only a general indication of the competence of the patient immune system, whereas the quantitative and functional assessment of virus-specific T-cell responses may be more relevant to patient’s risk stratification and clinical decision-making, thereby encouraging immune-monitoring of patients [13]. Moreover, while still experimental and often limited to research studies, adoptive immunotherapy with virus-specific lymphocytes could definitely benefit from more data on virus-specific immune-reconstitution to extend its applicability on a broader scale [13].

However, the implementation of data on virus-specific T-cell responses in the risk stratification and clinical decision-making of patients undergoing HSCT still needs validation in prospective clinical trials [14], and information is lacking about the current practice across centers. In September 2021, the Cellular Therapy & Immunobiology Working Party (CTIWP) of the European Society for Blood and Marrow Transplantation (EBMT) decided to conduct a survey across centers to identify current policies to monitor virus-specific immune reconstitution in patients undergoing allogeneic HSCT.

This study followed the EBMT study guidelines. All EBMT centers were invited to participate. A specific limited/closed questionnaire has been developed and sent to leaders of centers for completion directly or by delegates. Questions included the assessment of center practice regarding the monitoring of viral specific T-cell responses for CMV, HHV6 and AdV, asking details on the methodology of detection, the threshold levels of detection and the clinical use in specific transplant settings. The answers given by centers were summarized using descriptive statistics.

Policies for post-transplant virus-specific immune monitoring were collected for 152 EBMT centers, active in 37 countries (Fig. 1a), corresponding to 33% of the full reporting EBMT allogeneic sites. Centers have been classified as “smaller” (performing less than 50 allogeneic HSCT/year) or “larger” (performing at least 50 allogeneic HSCT/year) on the basis of their transplant volume per year [15]; consequently data on center activity have been showed according to this categorization (Fig. 1b). The participating centers perform allogeneic HSCT in adults only (43%), children only (26%) or both (31%), and use various donor sources (all centers use HLA-identical related, 94% matched unrelated, 99% mismatched related, 56% cord blood).

Fig. 1: Description of centers according to transplant volume and availability of immune reconstitution tests for CMV, HHV6 and AdV.
figure 1

a Distribution of centers by country and transplant volume per year (“smaller” or “larger” centers, performing < or ≥ 50 allogeneic HSCT/year respectively) [15]. b Distribution of different transplant types (MRD, MMRD, MUD, and UCB) within “smaller” and “larger” centers. Note that data on center volume was missing for 9% of centers in the survey, and therefore the proportions presented are among those with complete information. c Number of centers monitoring virus-specific T-cell responses for CMV, HHV6 and AdV in allo-HSCT recipients. Availability of tests in centers: clinical practice versus only experimental/clinical trials versus both clinical practice and experimental/clinical trials. AdV adenovirus, allo-HSCT allogeneic hematopoietic stem cell transplantation, CMV cytomegalovirus, HHV6 human herpes virus 6, MRD matched related donor, MMRD mismatched related donor, MUD matched unrelated donor, UCB umbilical cord blood.

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CMV viremia is routinely monitored after allogeneic HSCT in all centers, although 8 (5.3%) centers search for CMV only in case of seropositive donors and/or recipients. However, only 17 (11%) centers are currently testing CMV-specific T-cell responses, either within clinical practice (35%), experimental practice (12%), or both (53%) (Fig. 1c). Flow-cytometry based tests to assess CMV-specific T-cells (e.g. intracellular cytokines, MHC multimer binding), mainly set up in house, are used in 7 centers (41%). IFN-γ Enzyme-Linked Immunospot (ELISpot) is adopted in 15 (88%) centers, either with home-made or commercial assays. CMV Quantiferon is used in only 4 (24%) centers and mainly through commercial assays. Additionally, other home-made tests (e.g. proliferation assays or different CMV-specific T-cell subsets) are rarely applied. Thresholds are not harmonized among centers. In centers performing these assays, information on CMV-specific immune responses is currently taken into consideration to guide the treatment with “Off-the-Shelf” virus-specific T-cells and/or to decide on the duration of the antiviral therapy.

In 111 (73%) centers, AdV viremia was monitored in allogeneic HSCT recipients, in all HSCT recipients (59% of these centers), or in presence of potential risk factors (i.e. GvHD, aplastic anemia, alternative donor source, HLA-mismatched donor, in-vivo/ex-vivo T-cell depletion, second HSCT procedure). Moreover, 29% of the centers monitored the virus only in case of symptoms. Additionally, 8 centers check for AdV-specific T-cell responses, using different home-made or commercial assays (e.g. MHC multimer binding, intracellular cytokines, IFN-γ ELISpot, limiting dilution analysis), both as experimental and clinical practice (Fig. 1c). Main reasons to assess AdV-specific T-cell responses are: viral reactivation and/or low response of viremia to antiviral therapies.

Longitudinal monitoring for HHV6 viremia in allogeneic HSCT recipients is performed in 103 (68%) centers, in all HSCT recipients (53% of these centers) or in presence of potential risk factors (14%; including GvHD, previous viral reactivations, alternative donor source, HLA-mismatched unrelated donor, in-vivo/ ex-vivo T-cell depletion). Moreover, additional 33% of the centers search for the virus in case of clinical suspicion. Tests for HHV6-specific T-cell responses are reported by two centers (Fig. 1c), adopting home-made IFN-γ ELISpot or limiting dilution analysis for antigen-specific T-cells. One center performs the test in any case of HHV6 reactivation, while the other only in case of HHV6-related organ involvement.

Frequencies of T-cells specific for other viruses are currently tested in 15 (9.9%) centers, and in particular regarding Epstein-Barr virus (EBV; n = 14), BK virus (n = 4), varicella-zoster virus (VZV; n = 3), herpes simplex virus (HSV; n = 2) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; n = 1).

A higher proportion of ‘pediatrics only’ centers were smaller in terms of transplant volume (86% performing less than 50 allogeneic HSCT/year), as compared to ‘adults only’ (54%) or ‘adults/pediatrics’ (49%) centers. Additionally, ‘pediatrics only’ centers monitored AdV more frequently (87%) compared to ‘adults only’ (63%) or ‘adults/pediatrics’ (77%).

No relevant differences have been observed in monitoring policies among larger and smaller centers.

Overall, 21 (13.8%) centers are performing at least one type of virus-specific immune monitoring, while 14 (9.2%) centers at least two types (mainly CMV and AdV), 6 (3.9%) centers at least three types, and one center more than three types.

Furthermore, 47 (31%) centers are planning to start monitoring for virus-specific immune responses in the future, mainly for CMV (n = 41), HHV6 (n = 18), AdV (n = 19), EBV (n = 26), BK virus (n = 5), SARS-CoV-2 (n = 4).

The timely reconstitution, both for numbers and function, of a donor-derived immune system is of utmost importance for the recovery of wellbeing and long-term survival of patients after allogeneic HSCT [12]. According to this EBMT survey, immune monitoring for virus-specific T-cell responses is currently performed in a limited number of centers and is highly variable in terms of targets, technologies and interpretation. These results underline the need to harmonize and standardize methods, both for routine and investigational purposes. The heterogeneity of the adopted assays prevents us from detecting any specific trends. Accreditation in compliance with quality standards may be an additional requirement for a larger use of these functional tests and their comparability.

Moreover, the monitoring of CMV-specific responses should probably be more widely adopted for its clinical value in the new era of letermovir prophylaxis [16]. Since letermovir use potentially delays the occurrence of CMV infection and CMV-specific immune reconstitution, immunological [17] and virologic monitoring should be implemented after discontinuation of its prophylaxis. More frequent HHV6 [10] and AdV [11] cases have been reported across different donor sources and also in an adult setting with PT-Cy approaches [2, 3], potentially explaining this trend towards a more careful monitoring of viremia for many centers in recent years. In this context new monitoring guidelines are warranted.

In conclusion, improved reporting and communication between centers adopting these technologies is needed to foster collaborative and comparative research studies in the future, which may translate into new immune monitoring guidelines.