Antithymocyte globulins (ATG) cause profound T-cell depletion by targeting multiple surface antigens. Their main mechanisms include complement-dependent lysis, apoptosis, and opsonization with phagocytosis. Beyond depletion, ATG modulate immune responses by downregulating adhesion molecules and chemokine receptors, disrupting leukocyte trafficking [5]. ATG induces rapid and profound depletion of naive CD4⁺ T cells, more pronounced than that observed for CD8⁺ T cells. Notably, CD4⁺ lymphopenia often persists at 12 months post-transplantation [6].

ATG has been widely associated with profound immunosuppression and an increased incidence of cytomegalovirus (CMV) infection [7–11]. Since its initial use, it has been recognized that ATG may promote CMV infection by depleting CMV-specific cell-mediated immunity (CMI) [12].

According to the prospective multicenter observational study by Kumar et al., very few D+R- SOTRs exhibit detectable CMV-specific CMI, as measured by ELISPOT assay, at the time of transplantation [13]. However, this finding has been challenged by a single-center Spanish cohort study, which reported that approximately 25% of D⁺/R⁻ patients had detectable CMV-specific CMI at transplantation [14]. These differences may, at least in part, be explained by differences in CMV prevalence worldwide. Furthermore, most CMV-seronegative recipients do not develop a detectable T-cell response during the prophylactic period [12]. Consequently, at the end of prophylaxis, only a small proportion of D⁺/R⁻ SOTRs exhibit CMV-specific T-cell CMI [13]. Specifically, only 12.1% of D+R− patients have a positive QuantiFERON-CMV assay at the end of prophylaxis [15], a number that can rise to 20.9% when measured by ELISPOT assay [16]. Following these immunological observations, it has been demonstrated that ATG induction therapy, compared to anti-IL-2 receptor antagonists (IL-2RA), did not confer an additional risk of CMV DNAemia or disease in D⁺/R⁻ kidney transplant recipients [17]. As the increased risk of CMV associated with ATG is probably related to its depleting effect on CMV-specific T lymphocytes, this risk does not increase in patients who mostly lack CMV-specific CMI. Nevertheless, this treatment may exacerbate infection severity or recurrence rate, a possibility that requires further investigation.

In CMV-seropositive recipients (R+), most of the SOTRs have a CMV-specific CMI both at the time of transplantation and at the end of prophylaxis but this response is highly heterogeneous between the patients [13–18]. Consistent with these findings, it has been demonstrated that ATG induction therapy, compared with IL-2RA, confers an additional risk of CMV DNAemia and disease exclusively in R+ kidney transplant recipients [17]. Specifically, ATG increased the risk of CMV DNAemia in R+ recipients with detectable pretransplant CMI while no difference was observed among R+ recipients lacking detectable CMI, highlighting the pivotal role of CMV-CMI in the prevention of CMV infection (Figure 1). Furthermore, longitudinal analysis of CMV-specific CMI kinetics revealed that R+CMI+ recipients receiving rATG experienced a more pronounced suppression of their pre-existing CMV-specific immunity during the first two months post-transplantation compared to those treated with anti–IL-2RA, whereas no significant impact was observed in R+ CMI- recipients.

The recovery of CMV-specific CMI after ATG has been studied in few studies. At one-month post-transplantation, approximately 50% of R+ SOTRs receiving ATG recover a detectable CMV-specific CMI, as measured by the QuantiFERON-CMV assay [19]. By 3 months post-transplant using ELISPOT, CMV-specific immune recovery is comparable between ATG-treated and non–ATG-treated patients [17], and among patients with detectable pretransplant CMV-specific CMI, immune responses recovered by 6 months post-transplant to levels similar to those observed prior to transplantation [18].

Taken together, these findings suggest that ATG transiently impairs CMV-specific T-cell responses in R+ SOTRs, while allowing for subsequent immune reconstitution over time [12]. The transient loss of CMV-specific CMI in these patients is highly correlated with the risk of CMV disease. Few data are available about ATG effect on γδ T cells and NK cells.

Belatacept, a costimulation blocker targeting the CD28–CD80/CD86 pathway, has demonstrated efficacy in preventing alloimmune responses while preserving renal function. Clinical trials showed that belatacept used from transplantation did not increase CMV risk compared to cyclosporine. In the BENEFIT and BENEFIT-EXT trials, CMV infection rates were similar (6%–9% and 11%–14%, respectively) between belatacept and cyclosporine groups [20, 21]. In a smaller trial, Mannon et al. [22] observed more CMV viremia in the belatacept arm (20.7%), but differences were not statistically significant. Similarly, Woodle et al. [23] found comparable CMV rates (∼11%) across tacrolimus and two belatacept regimens. Switching from Calcineurin inhibitors (CNIs) to belatacept did not also appear to increase CMV disease incidence. Budde et al. [24] and Divard et al. [25] reported no rise in CMV complications after conversion, even in high-risk patients.

On the opposite, several retrospective studies have highlighted an increased risk of CMV infection in patients receiving belatacept. Bertrand et al. reported a higher incidence of late-onset CMV disease, following conversion to belatacept [26]. A subsequent study by the same group confirmed that both CMV-seropositive (R⁺) and high-risk CMV D⁺/R⁻ recipients were affected [27]. Similarly, Chavarot et al. described an increased incidence of atypical, often severe CMV disease—sometimes with intestinal or ocular involvement—occurring several years after transplantation (median 43.6 months post-conversion) [28]. These infections were particularly common among elderly recipients (>70 years), patients with impaired graft function, or those who underwent late conversion to belatacept. Moreover, CMV infections in belatacept-treated recipients frequently relapse despite initial antiviral therapy, occasionally necessitating discontinuation of belatacept [29]. A U.S. cohort by Karadkhele and colleagues similarly found higher CMV disease rates in CMV high-risk patients on belatacept, reinforcing that this association is not restricted to specific geographic regions or clinical settings [30].

Xu et al. demonstrated that belatacept’s immunosuppressive effects are more pronounced in naïve T cells but attenuate as T cells mature [31]. Belatacept trough levels vary widely among kidney transplant recipients, ranging from 1.4 to 24.8 μg/L, with a mean of 8.4 ± 3.9 μg/L [32]. In vitro, belatacept demonstrates strong immunosuppressive effects on alloreactive T cells, with an IC50 of 4 ng/mL for IL-2 production [33]. However, CMV-specific T cells are less susceptible to belatacept’s antiproliferative effect compared to alloreactive T cells [31]. In vivo, CMV-specific CD4⁺ and CD8⁺ T-cell responses remain stable during the first year after switching to belatacept, with no significant decline in IFN-γ or TNF-α secretion [34] (Figure 2).

The immunopathogenesis of CMV reactivation under belatacept likely stems from its targeted blockade of the CD28⁻CD80/CD86 costimulatory pathway, which is essential for the activation of naïve T cells. Regarding antiviral function, belatacept reduces IFN-gamma production by 50% in peripheral blood mononuclear cells (PBMCs) stimulated with a CMV peptide pool [33]. This inhibition dampens the primary CMV-specific T cell response, a particularly critical defect in seronegative recipients of CMV-positive organs (D⁺/R⁻). Consistent with this, Kleiboeker et al. reported a case of failed CMV-specific cellular immunity development in a high-risk D⁺/R⁻ patient treated with belatacept in combination with low-dose tacrolimus, MPA, and steroids [35].

Belatacept does not impair the frequency of IL-2/TNF-α/IFN-γ triple-positive CD28⁻ memory CMV-specific T cells, indicating that cytokine production by these memory cells remains largely preserved [31]. However, beyond its effects on naïve T cells, belatacept may indirectly impair CMV-specific memory T cells that are CD28-independent. Under physiological conditions, CD80 interacts in cis with PD-L1 on antigen-presenting cells, thereby limiting PD-1/PD-L1 engagement. Because belatacept has a high binding affinity for CD80, it can disrupt CD80/PD-L1 heterodimers [36]. This displacement could release free PD-L1, enhancing trans interactions between PD-1 and PD-L1, and thereby promoting functional exhaustion of PD1-expressing CMV-specific CD8⁺ T cells [37]. CMV infection itself may further amplify this inhibitory axis. The viral UL146 gene product (vCXCL1) has been shown to upregulate PD-L1 expression on infected hepatic cells, thereby strengthening PD-1/PD-L1–mediated suppression [38]. Moreover, CD28 blockade alters downstream signaling balance between NFAT and AP-1, fostering TOX-driven transcriptional programs associated with T-cell exhaustion. Consistent with this mechanism, Long et al. demonstrated that abatacept—another CD28 pathway inhibitor—induced T-cell exhaustion, supporting a class effect of CD28 blockade [39]. Finally, rare human cases of inherited CD28 deficiency are characterized by susceptibility to chronic viral infections, providing genetic evidence of the central role of this pathway in antiviral immunity [40] (Figure 3).

Given the increased CMV risk observed under belatacept, several strategies have been proposed to optimize its use while minimizing infection. In a recent study by Del Bello et al., conversion from MPA to an mTOR inhibitor in belatacept-treated kidney transplant recipients during or after a first CMV episode, led to a marked reduction in CMV replication or recurrence rates. In this study, the incidence of CMV DNAemia was 0.072 per month of exposure in patients treated with belatacept–MPA, compared with 0.035 in patients treated with belatacept–mTOR inhibitor (p = 0.0002) [41]. High-risk populations—particularly D⁺/R⁻ recipients, elderly patients—could also benefit early initiation of antiviral prophylaxis or pre-emptive therapy for at least 6 months following the initiation of belatacept therapy [26]. In the future, combining belatacept with agents that preserve CMV-specific immunity may help maintain the delicate balance between graft protection and antiviral defense [42].

CNIs target specific cytoplasmic proteins—cyclophilins for cyclosporine and FK-binding proteins for tacrolimus—thereby inhibiting calcineurin, a key phosphatase involved in the activation of lymphocytes. This inhibition prevents the transcription of cytokines such as IL-2, TNF-α, and IFN-γ, which are critical for the immune response following alloantigen recognition.

CNIs remain the cornerstone of immunosuppressive therapy, and most CNI-sparing strategies have been compared to regimens based on mTOR inhibitors or belatacept. This makes it particularly challenging to isolate the specific effect of CNIs on the risk of CMV infection after transplantation. However, a few studies have attempted to assess the risk of CMV viremia and/or infection in KTRs undergoing CNI withdrawal compared to those maintained on CNI therapy. In the Cochrane meta-analysis by Karpe et al. [43], which reviewed 83 studies—including 30 that specifically reported on CMV-related disease incidence—a trend toward a protective effect of CNI withdrawal was observed. However, this trend did not reach statistical significance. Therefore, the clinical impact of CNI on CMV infection has not been definitively documented in clinical trials. Another important question is whether tacrolimus and cyclosporine have a similar impact on CMV infection. In the pivotal trials comparing the two CNIs, the incidence of CMV infection was found to be similar [44, 45]. In the SYMPHONY study, Ekberg et al. [46] reported CMV infection rates of 14.6% in the standard-dose cyclosporine group, 11% in the low-dose cyclosporine group, and 9.7% in the low-dose tacrolimus group. More recently, a retrospective comparison between the EVERCMV study and a post hoc analysis by Tedesco-Silva and colleagues showed comparable CMV DNAemia rates between patients treated with cyclosporine and those treated with tacrolimus, whether combined with everolimus (EVR) (39.2% vs. 43.3%) or with mycophenolic acid (MPA) (77.8% vs. 77.9%) [47, 48]. Additionally, the type of CNI (tacrolimus versus cyclosporine) was not associated with viral eradication in the VICTOR study [49]. These findings support the hypothesis that both the incidence and severity of CMV infection are largely independent of the type of CNI used.

CNIs have been shown to exhibit direct antiviral properties, including the ability to impair viral replication. A recent study demonstrated that CNIs exert antiproliferative effects on CMV in vitro, with a sixfold increase in anti-CMV activity when combined with MPA. However, these effects were observed at drug concentrations exceeding those typically achieved in vivo based on pharmacokinetic data [50].

Additionally, several studies have investigated the impact of CNI on CMV-specific lymphocyte responses. In a recent study by Krueger et al., low-dose tacrolimus (5 ng/mL) did not significantly reduce early activation of CD4⁺ and CD8⁺ T cells upon stimulation with CMV pp65, as measured by CD69 expression [51]. However, tacrolimus alone significantly suppressed IL-2 production by both CMV-specific CD4⁺ and CD8⁺ T cells. Tacrolimus showed high potency in suppressing IL-2, with an IC₅₀ of approximately 1 ng/mL [33]. However, this IL-2 inhibition had little impact on the proliferative capacity of CMV-specific T cells, suggesting that another cytokine such as IL-15 could act as a substitute [51]. Cyclosporin has been tested on the proliferation capacity of both conventional CMV-specific and γδ T cells. A decreased proliferation capacity was observed at doses as low as 25 ng/mL [52].

Tacrolimus also reduces the production of IFN-γ, a key antiviral cytokine that limits CMV replication and dissemination. This effect is particularly pronounced in the CD4⁺ CMV-specific T cell subset [51]. Additional studies have confirmed that CNIs also suppress IFN-γ production in virus-specific memory CD8⁺ T cells [53]. Even at very low concentrations (1.5 ng/mL), tacrolimus was shown to reduce IFN-γ production by 80%–90% in CMV-specific T cells stimulated with CMV peptide pools for 72 h [33]. Furthermore, tacrolimus inhibits T cell responses in both CMV-naive and CMV-immune individuals [31]. However, tacrolimus does not appear to significantly impair cytolytic function, as it does not reduce the expression of perforin or granulysin in CMV pp65-stimulated PBMC [51].

Collectively, these findings highlight the nuanced impact of tacrolimus on CMV-specific T cell responses—sparing early activation and cytolytic potential, while strongly suppressing key cytokines like IFN-γ, which is essential for antiviral control (Figure 2).

MPA acts as a noncompetitive, selective, and reversible inhibitor of inosine monophosphate dehydrogenase, the rate-limiting enzyme in de novo guanosine nucleotide synthesis [54]. Because lymphocytes depend exclusively on this pathway for purine synthesis, MPA profoundly inhibits T- and B-cell proliferation while sparing other cell types that can utilize salvage pathways.

In patients with lupus nephritis, MPA therapy showed no higher incidence of CMV infection compared to cyclophosphamide or azathioprine, both during induction and maintenance phases [55, 56]. The absence of CMV disease in these patients likely reflects the absence of allograft-derived viral reintroduction or post-transplant–specific “hits,” such as ischemia–reperfusion injury, which are known triggers of CMV infection [57]. In renal transplantation, early landmark trials found no significant increase in CMV infection with MPA/cyclosporine regimens compared with azathioprine or placebo [58–60]. Later, Miller et al. reported that MPA (1–2 g/day) in combination with tacrolimus did not result in higher CMV infection rates than azathioprine-based therapy [61]. In the VICTOR trial, the use of MPA had no significant influence on viral eradication [49]. More recent retrospective analyses confirm that MPA was not an independent risk factor for CMV recurrence or late CMV DNAemia [62, 63]. Together, these clinical data suggest that MPA use, per se, does not increase CMV replication risk.

Among immunosuppressive agents, MPA displays the most selective antiviral actions against CMV in vitro. In combination with tacrolimus, MPA demonstrated synergistic lethality against CMV in cell culture models, although these effects occurred at concentrations exceeding those achieved clinically [50].

MPA induces broad suppression of adaptive immunity by depleting CD4⁺, CD8⁺, and CD19⁺ lymphocytes, in part through apoptosis [54]. It downregulates CD25 expression, thereby impairing IL-2–driven T-cell activation and proliferation. In vitro, MPA decreases IFN-γ production, not by impairing cytokine synthesis per cell, but by reducing the total lymphocyte pool capable of activation. Consequently, the antiviral arm of the immune response may be dampened in proportion to overall lymphocyte depletion. Recent studies have examined the effects of MPA on CMV-specific T-cell responses. Krueger et al. reported that activation of CD4⁺ and CD8⁺ T cells by CMV pp65 antigen is not significantly affected by MPA alone [51]. MPA had high IC50 values at 3 ng/mL for IL-2. However, MPA strongly reduces the proliferation of purified CMV-specific T cells and γδ T cells in vitro [52], indicating a selective antiproliferative effect without full functional suppression. Indeed, Egli et al. showed that MPA induced a moderate reduction in IFN-γ production by CMV peptide–stimulated T cells [33], and Krueger et al. found no significant reduction in IFN-γ–producing CMV-specific T cells upon MPA exposure [51]. Moreover, MPA does not impair cytotoxic T-cell function, since Krueger et al. observed preserved perforin and granulysin production and intact cytolytic activity of CMV-specific T cells exposed to MPA (Figure 2) [51].

In conclusion, MPA has not been shown to independently increase CMV replication or recurrence in SOT recipients. However, it is frequently the first immunosuppressive agent to be reduced or discontinued during CMV infection, mainly because of its myelosuppressive effects, its strong inhibitory impact on T-cell proliferation critical for CMV control, and its contribution to the overall burden of immunosuppression. Reducing or withdrawing MPA may therefore help limit cytopenias and promote immune recovery during CMV infection.