Cytomegalovirus-specific T cell immunity reconstitution following allogeneic hematopoietic stem cell transplantation: a pilot stduy

doi:10.21203/rs.3.rs-3337345/v1

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Research Article

Cytomegalovirus-specific T cell immunity reconstitution following allogeneic hematopoietic stem cell transplantation: a pilot stduy

https://doi.org/10.21203/rs.3.rs-3337345/v1

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Purpose

Reactivation of cytomegalovirus (CMV) leads to significant morbidity and mortality following allogeneic hematopoietic stem cell transplantation (allo-HSCT). The reconstitution of CMV-specific T cells plays a crucial role in the antiviral response after allo-HSCT. However, the impact of CMV reactivation on the recovery of CMV-specific T cells in the early stages after allo-HSCT, particularly haploidentical HSCT, remains undisclosed.

Methods

We retrospectively examined CMV-specific T-cell recovery in 78 allo-HSCT recipients to assess the influence of clinically significant CMV infection (CS-CMVi) on CMV-specific T-cell restoration.

Results

Patients in CS-CMVi group displayed higher absolute quantities of CMV-specific IFN-γ⁺ T cells on day 30 (CD4⁺ T cells: 1.40 vs. 0.07 cells/µL, p = 0.02; CD8⁺ T cells: 1.64 vs. 0.15 cells/µL, p = 0.11), but lower counts on day 180 (CD4⁺ T cells: 1.06 vs. 5.95 cells/µL, p < 0.01; CD8⁺ T cells: 3.70 vs. 55.36 cells/µL, p = 0.04). Among patients receiving letermovir prophylaxis (LTV group), the recovery of CMV-specific CD8⁺ T cells was significantly delayed compared to those receiving preemptive therapy (PET group) from day 60. The LTV group was more likely to experience late-onset CMV reactivation if their absolute counts of polyfunctional CMV-specific CD4⁺ T cells or CD8⁺ T cells was below 2.01 (AUC = 0.78, p = 0.003) or 0.90 cells/µL (AUC = 0.89, p < 0.001).

Conclusions

In conclusion, our pilot study provides direct evidence that early episodes of CS-CMVi impair the recovery of CMV-specific T cells after allo-HSCT. Additionally, insufficient polyfunctional restoration would lead to late-onset CMV reactivation in LTV group.

Cytomegalovirus (CMV)

CMV-specific T cells

allogeneic hematopoietic stem cell transplantation (allo-HSCT)

haploidentical HSCT (HID HSCT)

late-onset CMV reactivation

Cytomegalovirus (CMV) infection is a significant and potentially life-threatening complication post allogeneic hematopoietic stem cell transplantation (allo-HSCT), particularly pronounced in haploidentical (HID) allo-HSCT(1, 2). Reports indicate that around 60–95% of CMV-seropositive recipients encounter CMV reactivation after allo-HSCT, typically within the first three months in the absence of effective prophylaxis, with 20–30% progressing to CMV disease, which has substantial mortality (3, 4). Furthermore, CMV reactivation is linked to increased susceptibility to secondary bacterial and fungal infections, graft-versus-host disease (GvHD), and overall mortality within the first-year post allo-HSCT(5). Over the past few decades, the occurrence of CMV disease has drastically reduced due to the introduction of prophylactic and preemptive strategies that include ganciclovir, valganciclovir, and foscarnet. Nevertheless, the efficacy of these antiviral agents has been counterbalanced by their toxicity, including myelosuppression and nephrotoxicity. On the other hand, universal prophylaxis could impede the recovery of CMV-specific T-cell reconstitution (6, 7).

Restoration of CMV-specific T cells is imperative for sustained control of CMV reactivation following allo-HSCT(8). CMV-specific CD4⁺ and CD8⁺ T cells, which secrete protective cytokines like interferon γ (IFN-γ), interleukin-2 (IL-2), and tumor necrosis factor-α (TNF-α), play a crucial role in managing CMV reactivation(9). Deterioration in CMV-specific T-cell restoration correlates with increased CMV reactivation, recurrent or refractory CMV reactivation, and CMV disease. Monitoring the restoration of CMV-specific T cells assists in identifying patients at a high risk of CMV reactivation and CMV disease. Recently, immunologic monitoring employing commercially available cytokine release assays for CMV has gained attention, facilitating an improved risk stratification for immunocompromised patients and guiding prophylactic treatment. These assays quantify CMV-specific T cell reconstitution by measuring the cytokines released from CD4⁺ and CD8⁺ T lymphocytes in response to CMV antigens. Information about a patient’s CMV immune status helps clinicians evaluate the likelihood of CMV reactivation and the patient’s ability to clear or manage an infection effectively (10). Allo-HSCT recipients who lack CMV-specific CD4⁺ and CD8⁺ T cells demonstrate an increased incidence of CMV reactivation, and reconstitution of T-cell responses is consistent with protection against CMV.

The objective of this study was to retrospectively assess the effects of clinically significant CMV infection (CS-CMVi) episodes on CMV-specific T cell reconstitution, and to define characteristics of CMV specific T cell immunity in patients with late-onset CMV reactivation based on real-world experience.

Study population

We retrospectively analyzed a consecutive cohort of 78 adult patients with hematologic malignancies who underwent allo-HSCT at Shanghai Ruijin Hospital between April 2021 and December 2022. The last follow-up was June 30, 2023. The study was adhered to the principles of the Declaration of Helsinki. The requirement for written informed consent was waived owing to the non-interventional and retrospective nature of the study.

Transplantation procedures

Details of the preconditioning regimen, GvHD prophylaxis and treatment, and infection prophylaxis have been reported previously (10, 11). In brief, calcineurin inhibitors alongside short-term methotrexate and mycophenolate mofetil formed the backbone of GvHD prophylaxis. Myeloablative conditioning (MAC) and reduced-intensity conditioning (RIC) were applied in 72 and 6 patients, respectively. The stem cell sources consisted of granulocyte stimulating factor mobilized peripheral blood stem cell grafts from HID-related donors (n = 60), HLA-matched related donors (n = 11), and HLA-matched unrelated donors (n = 7).

Monitoring CMV reactivation

All included patients were identified as high-risk for CMV reactivation due to CMV seropositive donors and recipients, HID HSCT, and in vivo T deplete(12). CMV reactivation was monitored weekly by evaluating plasma CMV DNA through real-time quantitative polymerase chain reaction (RT-qPCR) during the first three months after allo-HSCT, followed by bi-weekly monitoring until the sixth month. Patients were assessed on scheduled visits, and if they tested positive, subsequent monitoring was adjusted to a weekly basis. CMV DNAemia indicated the detection of any level of CMV DNA in plasma samples. CMV DNAemia with viral load ≥ 1000 IU/mL or ≥ 500 IU/mL in two consecutive tests required preemptive therapy. CS-CMVi included CMV DNAemia necessitating preemptive therapy and/or CMV end-organ disease. Preemptive therapy involved the initiation of ganciclovir or foscarnet until negative CMV DNA was confirmed in two consecutive tests. CMV DNAemia, CMV end-organ disease, refractory and/or recurrent reactivation, and CMV disease were diagnosed following established criteria(2). Letermovir prophylaxis was administered from day 1 after allo-HSCT until day 100 at a dose adjusted to calcineurin inhibitors.

Peripheral blood mononuclear cell isolation and stimulation

Peripheral blood mononuclear cells (PBMCs) were isolated through density centrifugation from heparinized peripheral blood samples at various time points after allo-HSCT. PBMCs were processed within 24 hours of collection and cryopreserved at − 80°C using a solution of 90% fetal bovine serum (HyClone, Thermo Fisher) and 10% dimethyl sulfoxide (DMSO; Sigma-Aldrich). On the experimental day, PBMCs were rapidly thawed at 37°C, suspended in RPMI-1640 (Gibco, Thermo Fisher) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 2 mmol/L l-glutamine (Gibco, Thermo Fisher), 10,000 IU/L penicillin (Sigma-Aldrich), and 10 mg/L streptomycin (Gibco, Thermo Fisher). PBMCs were plated at a density of 1 × 10⁶ cells per well in 96-well round-bottom plates and stimulated for 6 h at 37°C with 5% CO₂, along with costimulatory antibodies anti-CD28 (1 µg/mL; BioLegend), anti-CD49d (1 µg/mL; BioLegend), and Brefeldin A solution (10 µg/mL; BioLegend). PBMCs were stimulated with pooled CMV 15-mer peptide libraries (including immediate early-1 [IE-1], IE-2, and phosphoprotein 65 [pp65]) (each at 1 µg/mL; JPT Peptide Technologies) dissolved in DMSO. Anti-CD3 (0.5 µg/mL; BioLegend) served as nonspecific positive controls, while samples containing DMSO (the peptide diluent) served as negative controls. Notably, not all samples were stimulated with all antigens due to insufficient cell quantities; thus, stimulation with pooled CMV 15-mer peptide libraries was prioritized.

Intracellular cytokine staining

A modified eight-color intracellular cytokine staining assay, including four functional markers specific to CMV-specific T cells, was adopted from previously published methods(13). After stimulation, cells were collected, washed with phosphate-buffered saline (PBS), and incubated in PBS with Live/Dead Fixable Viability Dye Brilliant Violet 510 (BD Bioscience) for 15 min. Afterward, the cells were washed using FACS wash buffer (BD Biosciences) and incubated in the dark for 30 min with a fluorescent antibody combination comprising anti-CD14 fluorescein isothiocyanate (FITC, BD Biosciences), anti-CD4 APC/Cy7 (Biolegend), and anti-CD8 PerCP/Cy5.5 (Biolegend). Following a double wash with FACS wash buffer, the cells were permeabilized for 20 min using 1× Cytofix solution (Biolegend), washed twice with Cytoperm buffer (Biolegend), and subsequently stained with a fluorescent antibody mixture containing anti-IL-2 PE (Biolegend), anti-IFN-γ APC (Biolegend), anti-TNF-α Brilliant Violet 405 (Biolegend), and anti-macrophage inflammatory protein (MIP)-1β PE/Cy7 (BD Biosciences) in Cytoperm buffer in the dark for 30 min. Finally, the cells were washed with Cytoperm buffer for subsequent flow analysis.

Flow cytometry and analysis

Cell acquisition (median PBMC counts: 3,160,000 [range: 40,000 ~ 32,900,000]) was conducted using a CantoII flow cytometer (BD Biosciences) post-staining. All antibodies were optimized for best performance, and appropriate single-color compensation and fluorescence minus-one (FMO) controls were implemented. Data was analyzed using FlowJo software, version 7 (Flow Jo LLC, USA). An initial time gate was established to ensure stable sample collection. Cells within the “live” gate (Brilliant Violet 510^–) were constrained by the CD14– gate to exclude monocytes. Subsequently, size (FSC) and granularity (SSC) parameters were applied to identify lymphocytes. CD4⁺ and CD8⁺ T-cell subsets were then gated for cytokine expression (IFN-γ, TNF-α, MIP-1β, and IL-2) (Supplemental Fig. 1). Positive responses were defined as at least three times the DMSO response in the same sample(14).

Statistical analysis

Data were censored at the time of death or last available follow-up. χ² test or Fisher’s exact test was employed for different categorical variables between the two groups. Nonparametric tests (Mann-Whitney U test for two groups) were used for continuous variables. The cumulative incidences (CIs) of viral infection (CMV, EBV, et.) were calculated with death as a competing risk and compared using the log-rank (Mantel-Cox) test. Relapse was defined as recurrence of bone marrow (BM) blasts > 5%, reappearance of blasts in peripheral blood, development of extramedullary disease, or by the recurrence of pre-transplantation chromosomal abnormalities. Event-free survival (EFS) events were defined as measurable detectable disease (MRD) positivity, relapse, or death from any cause. Overall survival (OS) events were defined as death from any cause. Cumulative incidence of relapse (CIR) was compared between groups as described previously by Gray with estimates reported by Kalbfleisch and Prentice(15).The Kaplan–Meier estimator was used to calculate the probabilities of survival. A p-value < 0.05 was considered statistically significant. The analyses were performed using SPSS version 20.0 (IBM SPSS Statistics, USA). The ROC curve was estimated based on the median value, and the area under the curve (AUC) was calculated using Medcalc software.

Patients’ characteristics

The patients’ characteristics are summarized in Table 1. The median age of the 78 patients was 46 years (range: 15–69). Patients without death had a median follow-up time of 416 days (range: 78–814). The CI of CS-CMVi for the entire cohort was 46.21%, with a median occurrence time of 39 days (range: 16–83) after allo-HSCT. Based on whether patients developed CS-CMVi within the first 100 days, we divided the entire cohort into two groups: CS-CMVi and non-CS-CMVi groups. Both groups were comparable in terms of age, sex, hematopoietic cell transplantation-specific comorbidity index (HCT-CI), underlying hematologic diseases, and the incidences of aGvHD and cGvHD. However, they differed in the incidence of non-CMV herpesvirus (NCH) reactivation, with the CS-CMVi group experiencing more episodes of NCH reactivation (Table 1, 72.9% vs. 48.7%, p = 0.02; Supplemental Fig. 2A). The proportion of patients with Epstein-Barr virus (EBV) reactivation (EBV DNA > 1 × 10⁴ IU/mL) was 59.4% in the CS-CMVi group and 24.3% in the non-CS-CMVi group (p < 0.001, Table 1). CIs for EBV reactivation were 52.18% and 27.12% in the CS-CMVi and non-CS-CMVi groups, respectively (Supplemental Fig. 2B, p = 0.02). Two patients, each in the CS-CMVi (2/37, 5.4%) and non-CS-CMVi (2/41, 4.8%) groups, developed post-transplantation lymphoproliferative disease (PTLD). No significant differences were observed between the CS-CMVi and non-CMVi groups regarding the reactivation incidences of herpes simplex virus-1 (HSV1, 18.9% vs. 14.6%, p = 0.61), human herpesvirus 6B (HHV6B, 22.9% vs. 12.2%, p = 0.26), and varicella-zoster virus (VZV, 5.4% vs. 2.4%, p = 0.56) (Table 1).

Table 1

Baseline characteristics of patients in CS-CMVi and non-CS-CMVi groups
Characteristics	Total (n = 78)	CS-CMVi (n = 37)	non-CS-CMVi (n = 41)	P value
Median age, years (range)	46(15–69)	47 (15–61)	46 (16–69)	0.94
Gender, n(%)				0.82
Male	39(50)	18 (48.6)	21 (51.2)
Female	39(50)	19 (51.4)	20 (48.8)
Follow up duration in days, median (range)	416(54–814)	484(54–793)	358(246–814)	0.10
Underlying disease, n(%)				0.94
AML	49(62.9)	23 (62.1)	26 (63.5)
ALL	20(25.6)	9 (24.3)	11 (26.9)
MDS	5(6.4)	3 (8.1)	2 (4.8)
Others	4(5.1)	2 (5.4)	2 (4.8)
HCT-CI scores before allo-HSCT, n(%)				0.84
0 (low risk)	58(74.4)	26(70.2)	32(78.0)
1–2 (intermediate risk)	14(17.9)	8(21.7)	6(14.6)
≥ 3 (high risk)	6(7.7)	3(8.1)	3(7.4)
Conditioning, n(%)				0.32
MAC	72(92.3)	33(89.1)	39(95.2)
RIC	6(7.7)	4(10.9)	2(4.8)
HLA matching, n(%)				0.33
Mismatched related	60(76.9)	30(81.0)	30(73.1)
Matched related	11(14.1)	3(8.1)	8(19.5)
Matched unrelated	7(9.0)	4(10.9)	3(7.4)
Blood group disparity, n(%)				0.13
Matched	30(38.5)	18(48.7)	12(29.2)
Major mismatched	21(26.9)	11(29.7)	10(24.4)
Minor mismatched	17(21.7)	5(13.5)	12(29.2)
Major and minor mismatched	10(12.9)	3(8.1)	7(17.2)
MNC counts in graft, median (range, ×10⁸/kg)	12.4(4.8–25.1)	12.6(5.8–21.3)	12.3(4.8–25.1)	0.93
CD34⁺cell counts in graft, median (range, ×10⁶/kg)	8.7(2.9–15.4)	8.9(5.3–15.4)	8.9(2.9–13.6)	0.50
Median time from HSCT to neutrophil engraftment (range)	14(10–24)	14(10–24)	13(11–20)	0.66
Median time from HSCT to platelet engraftment (range)	13(10–27)	12(10–27)	13(11–25)	0.20
Acute GvHD, n(%)
Grade ⅠI -IV	17(21.8)	10(27.0)	7(17.0)	0.28
Grade ⅡI - Ⅳ	6(7.6)	4(10.9)	2((4.8)	0.98
Chronic GvHD, n(%)
Moderate to severe	10(12.8)	4(10.9)	6(14.6)	0.87
NCH reactivation, n(%)	47(60.2)	27(72.9)	20(48.7)	0.02
EBV	33(42.3)	23(59.4)	10(24.3)	< 0.01
HSV1	13(16.7)	7(18.9)	6(14.6)	0.61
HHV6B	13(16.7)	8(22.9)	5(12.2)	0.26
VZV	3(3.8)	2(5.4)	1(2.4)	0.56
2-year CIR (%)	15.92	19.41	9.49	0.51
2-year EFS (%)	76.09	74.68	78.69	0.86
2-year OS (%)	97.19	97.22	97.05	0.76

Reconstitution of CMV-specific T cells in CS-CMVi and non-CS-CMVi groups

A total of 126 blood samples were available for analysis, including 30, 21, 21, 21, 19, and 14 samples at day 30 (d30), d60, d90, d180, d270, and d365 post allo-HSCT, respectively. Five healthy donor samples were used as controls. CMV-specific CD4⁺ and CD8⁺ T-cell reconstitution was assessed through flow cytometric immunoassays for cytokine staining, as previously reported(13). The median counts of CMV-specific IFN-γ⁺CD4⁺ and IFN-γ⁺CD8⁺ T cells in healthy donors were 2.05 and 8.15 cells/µL, respectively. Patients with CMV-specific IFN-γ⁺CD4⁺ or IFN-γ⁺CD8⁺ T cells surpassing 2.05 and 8.15 cells/µL, respectively, were considered to have a protective CMV-specific response. Detectable CMV-specific CD4⁺ and CD8⁺ T-cell responses exhibited a growing frequency and absolute counts over time in both groups. In the CS-CMVi group, 50.0% and 71.5% of patients showed a CMV-specific response at d90 and d365 in the CMV-specific IFN-γ⁺CD4⁺ T-cell recovery assay (Fig. 1), respectively. In the non-CS-CMVi group, the corresponding percentages were 70.0% and 100%, respectively. In the CMV-specific IFN-γ⁺CD8⁺ T-cell recovery assay, 50.0% and 57.1% of patients in the CS-CMVi group and 60.0% and 100% in the non-CS-CMVi group, respectively, exhibited a CMV-specific response at d90 and d365 (Fig. 1).

The recoveries of CMV-specific CD4⁺ and CD8⁺ T cells varied between the groups. At d30, patients in the CS-CMVi group displayed higher absolute counts of CMV-specific IFN-γ⁺CD4⁺T and IFN-γ⁺CD8⁺T cells compared to those in the non-CS-CMVi group (IFN-γ⁺CD4⁺T: 1.40 vs. 0.07 cells/µL, p = 0.02; IFN-γ⁺CD8⁺T: 1.64 vs. 0.15 cells/µL, p = 0.11; Table 2). The reconstitution of CMV-specific CD8⁺ T cells outpaced that of CMV-specific CD4⁺ T cells (Supplemental Fig. 3).

Table 2

Recoveries of CMV-specific IFNγ⁺CD4⁺ and CD8⁺ T cells in CS-CMVi and non-CS-CMVi groups
Variables	CS-CMVi median (range)	non-CS-CMVi median (range)	P value
CMV-specific IFNγ⁺CD4⁺T cells (cells/µL)
d30	1.40(0-6.42)	0.07(0-2.08)	0.02
d60	0.50(0.03–10.62)	2.66(0.04–15.40)	0.19
d90	5.12(0.85–6.03)	5.94(0.37–14.02)	0.44
d180	1.06(0.16–8.83)	5.95(3.20–9.77)	< 0.01
d365	4.61(0.25–25.32)	6.86(2.26–27.08)	0.09
CMV-specific IFNγ⁺CD8⁺T cells (cells/µL)
d30	1.64(0.00-14.36)	0.15(0.00-4.57)	0.11
d60	5.15(0-34.05)	7.98(0-77.42)	0.31
d90	3.03(0.90-39.49)	20.66(0.05–83.36)	0.57
d180	3.70(0.58–6.42)	55.36(50.62-178.28)	0.04
d365	23.01(0.25-255.82)	99.54(6.61-345.65)	0.36

CMV-specific IFN-γ⁺ T-cell counts (CD4⁺ and CD8⁺ cells) in the CS-CMVi group at d180 were significantly lower than those in the non-CS-CMVi group (IFN-γ⁺CD4⁺T cells: 1.06 vs. 5.95 cells/µL, p < 0.01; IFN-γ⁺CD8⁺T cells: 3.70 vs. 55.36 cells/µL, p = 0.04, Table 2). However, no significant differences of the absolute counts of CMV-specific CD4⁺ and CD8⁺ T cells were observed between the groups at d365 (IFN-γ⁺CD4⁺T cells: 4.61 vs. 6.86 cells/µL, p = 0.09; IFN-γ⁺CD8⁺ T cells: 23.01 vs. 99.54 cells/µL, p = 0.36, Table 2). Collectively, the reconstitution of CMV-specific T cells was slower in patients experiencing a CS-CMVi episode within the initial 100 days, and then recovered to normal level within a year after allo-HSCT.

The recoveries of CMV-specific TNF-α⁺CD4⁺ T and TNF-α⁺CD8⁺ T cells mirrored the pattern of CMV-specific IFN-γ⁺ T cells (Supplemental Table 1). The recovery of CMV-specific MIP-1β⁺CD4⁺ T cells was slower compared to that of IFN-γ⁺CD4⁺ T cells, as MIP-1β⁺CD4⁺ T cells were scarcely detectable during the initial 180 days post allo-HSCT. The recovery of CMV-specific MIP-1β⁺CD8⁺ T cells closely resembled that of IFNγ⁺CD8⁺ T cells (Supplemental Table 2). CMV-specific IL2⁺ T cells were undetectable within the initial 180 days, as effector memory IL2⁺ T cells typically recover after 6 months following allo-HSCT(16) (Supplemental Table 3). These results indicated that early exposure to CMV antigens could trigger the expansion of specific T cell subsets, particularly IFN-γ⁺ and MIP-1β⁺ T cells. However, an early CS-CMVi episode led to delayed reconstitution across all CMV-specific T-cell subsets.

Furthermore, we explored the impact of CMV viral load on CMV-specific T-cell reconstitution. In comparison to patients with low CMV viral load (peak CMV DNA ≤ 1 × 10⁴ IU/mL), patients with high viral load (peak CMV DNA > 1 × 10⁴ IU/mL) exhibited significantly lower CMV-specific IFN-γ⁺ and TNF-α⁺ T cells at d60, though the gap was narrowed at d180 (Fig. 2A). In patients with CMV disease, reconstitution of both CMV-specific IFN-γ⁺ and TNF-α⁺ T cells was markedly delayed during the initial 60 days when compared to their counterparts in non-CS-CMVi group and patients with CMV DNAemia only, respectively (Fig. 2B). CMV-specific IFN-γ⁺CD4⁺ T cells were undetectable by d180 in the CMV disease group, suggesting a crucial role of IFN-γ⁺CD4⁺ T cell subset in immune defense against CMV.

The CIR at 2 years in patients with and without CS-CMVi was 19.41% (95% CI, 0.19–2.28) and 9.49% (95% CI, 0.43–5.22, p = 0.51, Table 1), respectively. No significant differences were found in both EFS and OS between the CS-CMVi and non-CS-CMVi groups (Table 1).

Reconstitution of CMV-specific T cells with different CMV treatment modalities

Before the introduction of letermovir in China (August 2022), allo-HSCT recipients who were predetermined to receive preemptive therapy in case of CMV reactivation were grouped as the PET group (n = 55). After that, allo-HSCT recipients who received letermovir prophylaxis were grouped as the LTV group (n = 23). A comparison of pretransplant characteristics and clinical findings between the two groups is presented in Table 3. During the initial 180 days, The CIs of any detected levels of CMV DNA were 70.9% and 39.1% for the PET and LTV groups, respectively (p < 0.01, Fig. 3A). The CIs of CMV DNAemia (≥ 500 IU/mL) were 67.2% and 30.4% for the PET and LTV groups, respectively (p < 0.001, Fig. 3B). Specifically, patients in the LTV group exhibited lower occurrences of CS-CMVi in the first 100 days (34.5% vs. 4.3%, p < 0.01), but a higher tendency of late-onset CMV reactivation after day 100 (26.0% [6/23] vs. 5.5% [3/55], p < 0.01, Table 3). A statistically significant higher CMV DNA peak load was observed in the PET group (7780 IU/mL [range, 630–48700]) compared to the LTV group (1000 IU/mL [range, 960–13400], p = 0.01, Table 3). Preemptive therapy was required by 65.4% of patients in the PET group and 4.3% of patients in the LTV group within the first 100 days (p < 0.01). Patients in the PET group underwent longer durations of preemptive therapy than their counterparts in the LTV group (median time: 17 days [range, 7–31] vs. 12 days [range, 7–22], p = 0.01, Table 3).

Table 3

Clinical characteristics of patients in PET and LTV groups
Characteristics	PET group (n = 55)	LTV group (n = 23)	P value
Median age, years (range)	40(15–61)	55(19–69)	< 0.01
Follow up duration in days, median (range)	482(54–814)	332(246–618)	< 0.01
Gender, n(%)			0.80
Male	27(49.1)	12(52.1)
Female	28(50.9)	11(47.9)
HLA matching, n(%)			< 0.01
Mismatched related	31(56.3)	22(95.6)
Matched related	18(32.7)	0(0)
Matched unrelated	6(11.0)	1(4.4)
Conditioning, n (%)			0.17
MAC	51(92.7)	19(82.6)
RIC	4(7.3)	4(17.4)
Acute GvHD, n(%)
Grades Ⅱ - Ⅳ	11(20.0)	6(26.0)	0.55
Grades ⅡI - Ⅳ	4(7.2)	2(8.8)	0.82
Chronic GvHD, n(%)
Moderate to severe	8(14.5)	2(8.8)	0.48
CMV infection, n(%)
CMV DNAemia by day 100	36(34.5)	1(4.3)	< 0.01
CMV DNAemia after day 100	3(5.5)	6(26.0)	< 0.01
CMV disease by day 100	4(7.3)	0	0.18
CMV disease after day 100	6(10.9)	3(13.0)	0.76
R/R CMV infection	6(10.9)	1(4.3)	0.35
Peak CMV DNAemia (×10³ IU/mL), median (range)	7.78(0.63–48.7)	1.00(0.96–13.4)	0.01
CMV duration, median (range, days)	17(7–31)	12(7–22)	0.01
NCH reactivation, n(%)	34(61.8)	13(56.5)	0.66
EBV	27(44.1)	6(26.1)	0.16
HSV1	8(14.5)	5(23.8)	0.43
HHV-6B	9(16.3)	4(17.3)	0.90
VZV	3(5.4)	0	0.25
1-year CIR (%)	9.90	12.50	0.51
1-year EFS (%)	87.06	79.16	0.43
1-year OS (%)	96.13	91.66	0.35

No significant differences were found in the CIs of CMV disease within the first year between the two groups (PET vs. LTV: 9.25% vs. 13.04%, p = 0.65, Fig. 3C). During the initial 100 days, four patients developed CMV disease in the PET group (7.3%) (pneumonia [n = 1], gastroenteritis [n = 3]) at a median time of 41 days (range, 28–55). No cases of CMV disease were diagnosed in the LTV group during the same time period. Late-onset CMV disease occurred in six patients in the PET group (pneumonia [n = 2], central nervous system [n = 1], retinitis [n = 3]) and three patients in the LTV group (pneumonia [n = 2], gastroenteritis [n = 1]) at median time of 124 days (range, 108–254) and 134 days (range, 114–160), respectively (p = 0.76). Among patients with CMV disease, 92.3% (12/13) were diagnosed antecedent CMV DNAemia with an interval of 7 days (range, 0–126), except for one patient in the PET group who developed late-onset CMV retinitis without preceding CMV DNAemia.

The CIs of NCH infection were 62.59% in the PET group and 56.52% in the LTV group (p = 0.95, Supplemental Fig. 2C). The CIs of EBV reactivation in the PET and LTV groups were 44.47% and 26.09%, respectively (p = 0.16, Supplemental Fig. 2D). No significant difference was found in the incidence of HSV1, HHV-6B or VZV reactivation between the two groups (Table 3).

One-year CIR in the PET and LTV groups were 9.90% (95%CI, 0.14–2.93) and 12.50% (95%CI, 0.34–6.82, p = 0.51, Table 3), respectively. The survival outcomes were equivalent between the two groups (Table 3).

As summarized in Table 4, the PET group exhibited a higher count of CMV-specific CD4⁺ and CD8⁺ T cells compared to those in the LTV group within the initial 60 days, which were consistent with the results reported by Zamora et al. (13) and Camargo et al. (17) (Table 4, Supplemental Table 4–6). The recovery of CMV-specific IFN-γ⁺ and TNF-α⁺ CD8⁺ T cells was significantly delayed in patients in the LTV group compared to those in the PET group (IFN-γ⁺CD8⁺T cells: 0.21 vs. 11.50cells/µL, p = 0.04, Table 4; TNF-α⁺CD8⁺T cells: 0.27 vs. 10.31 cells/µL, p = 0.04, Supplemental Table 4). Similar trends were observed for CMV-specific IL2⁺ and MIP-1β⁺ T cells (Supplemental Table 3, 5). In conclusion, the reconstitution of CMV-specific CD8⁺ T cells in the LTV group was markedly delayed from d60 after HSCT.

Table 4

CMV-specific IFNγ⁺CD4⁺ and CD8⁺ T cells in PET and LTV groups
	PET group median (range)	LTV group median (range)	P value
CMV-specific IFNγ⁺ CD4⁺ T cells (cells/µL)
d30	0.45(0-4.30)	0.17(0-6.42)	0.91
d60	1.94(0.03–10.62)	0.82(0.04–4.14)	0.46
d90	5.97(0.37–14.02)	3.56(0.50–12.40)	0.62
CMV-specific IFNγ⁺CD8⁺T cells (cells/µL)
d30	0.67(0-14.36)	0.07(0-4.88)	0.29
d60	11.55(0.02–77.42)	0.21(0-14.93)	0.04
d90	26.65(2.19-139.49)	1.07(0.05–58.97)	0.02

Reconstitution of polyfunctional CMV-specific T cells

T-cell reconstitution plays a pivotal role in antiviral responses. However, functional T cell reconstitution was not always synchronized with peripheral blood T cell subsets. To study the underlying relationship between T cell immune reconstitution and CMV reactivation, we first investigated the recovery of absolute counts of CD4⁺ and CD8⁺ T cells in peripheral blood. Total lymphocytes, CD4⁺, and CD8⁺ T cells did not differ significantly between the CS-CMVi and non-CS-CMVi groups at d30, d60, d90, d180, and d365, respectively (Supplemental Table 7). In a subgroup analysis, patients with recurrent and/or refractory CMV reactivation exhibited notably lower CD8⁺ T cell counts at d60 (27 cells/µL vs. 199 cells/µL, p = 0.03, Supplemental Fig. 4) compared to those without.

We defined polyfunctional CMV-specific T cells as those expressing IFN-γ in combination with other positively labeled markers (IL2, MIP-1β, or TNF-α). The proportion of polyfunctional T cells was compared longitudinally in Fig. 4A. Polyfunctional CD4⁺ and CD8⁺ T cell responses peaked at d90 in both CS-CMVi and non-CS-CMVi groups, and the occurrence of CS-CMVi slightly suppressed polyfunctional CD4⁺ and CD8⁺ T cell responses at d180. The absolute counts of functional CMV-specific T cells after combining IL2⁺, MIP-1β⁺, and TNF-α⁺ CD4⁺ or CD8⁺ T cells in CS-CMVi and non-CS-CMVi groups were shown in Table 5. Early CMV reactivation led to increased counts of functional CD4⁺ and CD8⁺ T cells within the initial 90 days, while they were diminished by d180. In summary, the results indicated that early CMV reactivation may enhance the polyfunctional responses of CMV-specific T cells within three months but impede the polyfunctional recovery of CMV-specific T cells in the long term.

Table 5

CMV-specific Polyfunctional T cells in CS-CMVi and non-CS-CMVi groups
	CS-CMVi Median (range)	Non-CS-CMVi Median (range)	P value
CMV-specific polyfunctional CD4⁺ T cells (cells/µL)
d30	2.40(0.00-25.29)	1.90(0.01–6.05)	0.714
d60	2.88(0.03–10.62)	3.68(0.04–15.40)	0.12
d90	7.23(0.85–12.40)	1.73(0.37–14.02)	0.38
d180	3.22(0.16–8.83)	9.00(3.20–9.77)	0.15
CMV-specific polyfunctional CD8⁺T cells (cells/µL)
d30	2.95(0.00-14.36)	0.43(0.00-4.57)	0.31
d60	5.27(0-34.05)	16.71(0-77.42)	0.31
d90	69.64(2.19-139.29)	12.23(0.05–83.36)	0.16
d180	2.82(0.58–6.42)	28.33(50.62-178.28)	0.017

Reconstitution of CMV-specific T cells in patients with late-onset CMV reactivation

Among patients in the LTV group, six developed late-onset CMV reactivation after discontinuation of letermovir. These patients exhibited lower absolute counts of CMV-specific IFN-γ⁺CD4⁺ and IFN-γ⁺CD8⁺ T cells at d60 compared to those who did not (IFN-γ⁺CD4⁺T: 0.88 vs. 0.07 cells/µL, p = 0.01; IFN-γ⁺CD8⁺: 3.08 vs. 0.05 cells/µL, p = 0.01, Supplemental Fig. 5). The recovery of other subtypes of CMV-specific CD4⁺ and CD8⁺ T cells, as well as polyfunctional CMV-specific CD4⁺ and CD8⁺ T cells, was delayed in patients with late-onset CMV reactivation (polyfunctional CD4⁺T cells: 0.55 cells/µL vs. 2.04 cells/µL, p = 0.019; polyfunctional CD8⁺ T cells: 0.12 cells/µL vs 8.72 cells/µL, p = 0.019, Fig. 4B and 4C).

We conducted an ROC analysis to determine the predictive capacity of polyfunctional CMV-specific T cells at d60 for late-onset CMV reactivation after letermovir discontinuation. The analysis revealed that CMV-specific polyfunctional CD4⁺ T cell counts > 2.01 cells/µL (AUC = 0.78, p = 0.003) or CD8⁺ T cell counts > 0.90 cells/µL (AUC = 0.89, p < 0.001, Fig. 5) was associated with a protective effect against late-onset CMV reactivation. In the LTV group, late-onset CMV reactivation occurred in 60% patients (6/10) of whom the absolute number of polyfunctional CMV-specific CD4⁺ T cells at d60 was < 2.01 cells/µL. In contrast, only 9.1% of patients (1/11) over that threshold experienced late-onset CMV reactivation. Similarly, in respective of the polyfunctional CMV-specific CD8⁺ T cell subset, late-onset CMV reactivation occurred in 60% patients (6/10) of whom the absolute count of polyfunctional CMV-specific CD8⁺ T cell at d60 was < 0.90 cells/µL, instead, none of patients above the threshold developed late-onset CMV reactivation.

This study investigates the dynamics of CMV-specific T cell reconstitution after allo-HSCT in patients with and without CS-CMVi. All patients included in this study had a CMV serostatus of D+/R+, with 76.9% (60/78) were being HID HSCT, which might lead to different reconstitution process of CMV immunity.

The reconstitution of CMV-specific T cells begins with the peripheral expansion of antigen-experienced memory T cells, which subsequently multiply upon encountering the antigen(18). Comparable to prior observations, the recovery of total CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells followed similar patterns in both CS-CMVi and non-CS-CMVi groups(19). Thus, our focused on enumerating CMV-specific CD4⁺ and CD8⁺ T cells, as these subsets of functional T cells have been shown to offer protection against CMV reactivation within the first year following transplantation(20, 21). As shown in our results, CMV reactivation prompted the expansion of CMV-specific CD4⁺ T and CD8⁺ T subsets during the initial 60 days. This observation is in line with previous findings that documented how subclinical CMV reactivation with ganciclovir prophylaxis might facilitate the functional recovery of T cells (6, 7). Our results also confirm the deleterious influence of high CMV viral load and CMV disease on the reconstitution of CMV-specific T cell immunity.

Over the past decade, significant progresses have been made in understanding the immune control system during CMV exposure following allo-HSCT(22–25). Earlier studies suggested that the reconstitution of CD4⁺ T cells precedes and subsequently orchestrates CD8⁺T cell reconstitution to provide enhanced protection against CMV disease(26, 27). CMV-specific CD4⁺ T cells have been reported to be indispensable for maintaining CMV-specific CD8⁺ T cell levels that ensure long-term viral control(27, 28). In asymptomatic individuals with CMV reactivation, the CMV-specific CD4⁺ T cell response preceded CMV-specific CD8⁺ T-cell responses, and in symptomatic individuals, the CMV-specific effector memory CD4⁺ T-cell response was delayed and detectable only after antiviral therapy (26). Our study found that the undetectable IFN-γ⁺CD4⁺ T cells at d180 was linked to CMV disease. However, the robust acquisition of detectable and protective polyfunctionality among CMV-specific CD4⁺ and CD8⁺ T cells seemed to occur more rapidly in patients without CS-CMVi during the initial 90 days (Table 4). This finding is consistent with documented cases wherein polyfunctional T cells appear more effective than monofunctional T cells in controlling CMV replication(29, 30).

CMV-specific IL2⁺ T cells are rarely detected during the initial 30 days (26). IL-2 production is vital for sustaining T cell expansion and is generated after the initial T cell priming, as well as by central memory T cells. Conversely, an IFN-γ⁺IL2⁺ cytokine profile typically characterizes cells exhibiting an effector memory phenotype(31). Beyond 180 days, over half of all CMV-specific CD4⁺ T cells produces IL-2, a pattern similar to that seen in healthy donors. Our results are in line with findings from Jasper J. P. van Beek’s group, which found that CMV-specific CD8⁺ T cells rarely express IL-2 compared to CMV-specific CD4⁺ T cells(32). Yet, IFN-γ, MIP-1β, and TNF-α producing CMV-specific CD4⁺ T and CD8⁺ T cells all exhibited prompt responses in early CMV reactivation after allo-HSCT. An arrangement of IFN-γ⁺IL2⁺MIP-1β⁺TNF-α⁺ was identified as a protective signature of CMV-specific CD8⁺ T cells(17). We found that the response of CMV-specific IFN-γ⁺IL2⁺MIP-1β⁺TNF-α⁺ T cells peaked at d90, and a negative correlation was observed between the recovery of the subset within the initial 90 days and early episode of CMV reactivation.

As the global management of CMV has transformed since the FDA approved of letermovir(3, 33, 34), it becomes vital to in-depth comprehend the impact of letermovir prophylaxis on CMV-specific T-cell reconstitution. Earlier investigations have indicated reduced levels of polyfunctional CMV IE1 and pp65-specific T cells at d90 with letermovir prophylaxis(13). Impaired reconstitution of CMV-specific CD4 and CD8 T cells is associated with an increased risk of late-onset CMV reactivation(35). In the phase III study that led to letermovir’s approval for the prophylaxis of CMV-seropositive allo-HSCT recipients(33), the incidence of CMV reactivation started to rise in the letermovir group around week 18. This suggests a delayed onset of CMV reactivation, a trend mirrored in our study. The reduced monitoring frequency beyond day 100 may have postponed the initiation of preemptive therapy, potentially contributing to severe late-onset CMV disease, if not, in patients with poor immune reconstitution. In our study, letermovir could delay CMV-specific T cell recovery as early as 60 days after allo-HSCT. We defined CMV-specific polyfunctional CD4⁺ T cells < 2.01 cells/µL or CD8⁺ T cells < 0.90 cells/µL at d60 as high-risk indicators for late-onset CMV reactivation in the LTV group. Continual routine monitoring of CMV DNA or an extended prophylactic approach could serve as important protective measures for patients with insufficient polyfunctional CMV immunity reconstitution. Recent research has proposed the potential benefit of extended letermovir prophylaxis until d180, which effectively diminished the incidence of CS-CMVi between d100 and d180, a time frame during which letermovir was withdrawn (36). However, further research is necessary to determine the time frame for letermovir prophylaxis in different transplant modalities.

Our study has several limitations due to its retrospective design, small sample size, and relatively short follow-up duration. Although CMV reactivation is a significant contributor to non-relapse mortality in allo-HSCT, numerous studies have also highlighted a protective aspect of CMV reactivation against relapse in AML and ALL(37–39). Owing to the small cohort and limited follow-up, we could not evaluate the protective effect of CMV reactivation against relapse. The intricate interplay between GvHD (both acute and chronic) and CMV reactivation also warrants further investigation. Thus, larger multicenter retrospective studies or prospective research endeavors are needed.

In conclusion, our findings suggest that CMV exposure might improve the recovery of CMV-specific T cells in the initial period of allo-HSCT. However, CMV reactivation tends to delay CMV-specific immune reconstitution over the long term. Letermovir prophylaxis would delay the recovery of CMV-specific T cells, especially CD8⁺ T cells from d60, potentially increasing the incidences of late-onset CMV reactivation upon letermovir discontinuation. CMV-specific polyfunctional CD4⁺ T cells > 2.01 cells/µL or CD8⁺ T cells > 0.90 cells/µL exhibits a protective effect against late-onset CMV reactivation in letermovir prophylaxis.

Funding

This work was supported by the National Key Research and Development Program of China (2022YFC2502600 to X.H.), the National Natural Science Foundation of China (82170206 to X.H., 82000156 to L.W.), Shanghai Municipal Health Commission Project of Disciplines of Excellence (20234Z0002 to X.H.) and Shanghai Sailing Program grant (20YF1439700 to L.W.).

Declaration of interests

The authors declare no competing interests.

Availability of data and material

The data underlying this article are available in the article and in its online supplementary material.

Contributions

L.W. and S.L. processed patient samples, performed in vitro experiments, collected clinical data and wrote the manuscript. J.H., Z.Z., Z.P., C.J., and S.Z collected samples, treated patients, and interpreted the data. X.C., G.C. and X.H. proposed and designed the study, interpreted the data, wrote the manuscript, and oversaw the project.

Ethics Approval

The study was approved by the Ethics Committee of Ruijin hospital.

Consent to Participate

The requirement for written informed consent was waived owing to the non-interventional and retrospective nature of the study.

Consent for Publication

Not applicable.

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Cytomegalovirus-specific T cell immunity reconstitution following allogeneic hematopoietic stem cell transplantation: a pilot stduy

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