CMV infection combined with acute GVHD associated with poor CD8+ T-cell immune reconstitution and poor prognosis post-HLA-matched allo-HSCT

Abstract

Cytomegalovirus (CMV) infection and acute graft-versus-host disease (aGVHD) are two major complications that contribute to a poor prognosis after hematopoietic stem cell transplantation (HSCT). Superior early immune reconstitution (IR) is associated with improved survival after HSCT. However, when all three factors, CMV infection, aGVHD, and IR, are concomitantly considered, the effects of the triple events on HSCT are still unknown and should be studied further. Thus we enrolled 185 patients who were diagnosed as hematological malignancies and treated with HLA-matched sibling transplantation (MST) between January 2010 and December 2014, of whom 83 were positive for CMV infection and 82 had aGVHD. Results showed that patients with both aGVHD and CMV infection had significantly higher non-relapse mortality (NRM), lower overall survival (OS), and delayed CD8+ T-cell IR. Multivariate analyses showed that both aGVHD combined with CMV infection and delayed CD8+ T-cell IR were independent risk factors for prognosis post-MST. Recurrent CMV infections are associated with poor CD8+ T-cell reconstitution. However, superior IR could protect against the negative effects of aGVHD and CMV infection on the transplant outcomes.

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Introduction

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the most effective treatment for patients with hematological malignancies or life-threatening non-malignant hematological diseases. However, allo-HSCT success is negatively affected by non-relapse mortality (NRM) and relapse. Acute graft-versus-host disease (aGVHD) and cytomegalovirus (CMV) infections are extremely common complications that lead to poor prognosis [1–5].

Poor immune reconstitution (IR) is also associated with adverse outcomes post-transplantation. Lymphocytes, CD4+ T cells, CD8+ T cells, and NK cells have been demonstrated to be associated with transplant outcomes, and early reconstitution of these cells has also been used as a predictor of transplant outcomes in different studies [6–9], further, B cells also affect transplant outcomes because of its important role in humoral immunity reestablishment after transplantation [10, 11]. Researchers have studied the relationship between CMV infection and aGVHD, CMV infection and IR, aGVHD and IR, and results showed that CMV infection and aGVHD are risk factors for each other [12, 13], poor IR is highly associated with CMV infection [3], Tregs in the PB after allo-HSCT may protect from aGVHD [14], functional dysregulation of B cells correlated with aGVHD and CMV infection [10, 11], and aGVHD can influence the bone marrow niche and IR [4]. However, when all three factors, CMV infection, aGVHD, and IR, are concomitantly considered, the effects of the triple events on HSCT are still unknown and should be studied further.

In this study, we evaluated the relationship between CMV infection, aGVHD, and IR, and revealed their effects on transplant outcomes, as well as the association between IR and outcomes after the onset of aGVHD and CMV infection in an adult HLA-matched sibling HSCT cohort.

Materials and methods

Study design

This study was performed at Peking University People’s Hospital and approved by the Ethics Committee of Peking University People’s Hospital. A total of 185 patients diagnosed with hematologic malignancies who underwent HLA-matched sibling allo-HSCT (MST) between January 2010 and December 2014 were retrospectively enrolled.

Transplants

All patients received a myeloablative conditioning regimen (MAC) without in vitro T cell depletion. The conditioning regimen used in our center included BU-based or total-body irradiation (TBI). Patients undergoing MST received hydroxycarbamide (80 mg/kg) orally on day −10 and a lower dose of cytarabine (2 g/m² per day) on day −9, busulfan (3.2 mg/kg per day, intravenously on days −8 to −6), cyclophosphamide (CY, 1.8 g/m² per day) intravenously on days −5 to −4, and Me-CCNU (250 mg/m²) orally once on day −3. The TBI-based conditioning regimen was composed of TBI (770 cGy for a single dose) on days −6 and cyclophosphamide (CY, 1.8 g/m² per day) intravenously on days −5 to −4; Me-CCNU (250 mg/m²) orally once on day −3.

GVHD prophylaxis and treatment

All transplant recipients received cyclosporine A (CsA), mycophenolate mofetil (MMF), and short-term methotrexate (MTX) for GVHD prophylaxis. CsA was administered intravenously on day −9 at a dosage of 2.5 mg/kg, which was adjusted to a blood concentration of 150–250 ng/ml. CsA treatment was gradually reduced and discontinued at approximately 4–6 months after HCT. MTX (15 mg/m²) was administered intravenously on day +1, and 10 mg/m² was administered on days +3 and +6 to patients receiving MST. MMF was administered orally (0.5 g every 12 h) from day −9 but was discontinued upon engraftment. Acute GVHD was treated with steroids (methylprednisolone, 1 mg/kg/day) as the first-line therapy. Anti-CD25 monoclonal antibodies (basiliximab; Novartis Pharma AG, Basel, Switzerland) were administered as a second-line therapy [11]. Chronic GVHD (cGVHD) was treated using CsA, mycophenolate mofetil, or steroids.

CMV surveillance

CMV reactivation was monitored using DNA quantitative polymerase chain reaction (Q-PCR) in plasma samples collected twice weekly for up to 100 days post-SCT. The time to CMV reactivation was defined as the time from the day of transplantation to the first CMV-positive Q-PCR result of >1000 gc/ml [15].

Immune reconstitution monitoring

Absolute monocyte and lymphocyte counts were determined by routine blood examination in the clinical hematology laboratory 30, 60, and 90 days after transplantation. Immune cell subsets (CD3+ T, CD3+CD4+ T, CD3+CD8+ T, and CD19+ B cells) were identified using multiparameter flow cytometry, as previously published studies [16].

Statistical analyses

The two groups were compared using the χ² test or Fisher’s exact test for categorical variables and the Mann–Whitney U test for continuous variables. Univariate and multivariate analyses of time-dependent outcomes were performed using Kaplan–Meier estimates and Cox proportional hazard models, and logistic regression was used for multivariate analysis of categorical variables. Kendall’s tau-b analysis was used to analyze the correlation between aGVHD and CMV infection. Unless otherwise specified, P-values were based on two-sided hypothesis tests, and α was set at 0.05. Most analyses were performed using SPSS software (version 22.0; Chicago, IL, USA). Final follow-up was conducted on 19 February 2020.

Results

Patients’ characteristics

A total of 185 patients diagnosed with hematological malignancies who underwent MST between January 2010 and December 2014 were retrospectively enrolled in this study. The patient characteristics are listed in Table 1. The median age of the patients was 41 years (range, 2–66 years). Patients with primary diseases included those with acute lymphoblastic leukemia (n=51, 27.6%), acute myeloid leukemia (n=80, 43.2%), and myelodysplastic syndrome (n=29, 15.7%). The incidence rates of CMV infection and acute GVHD were 44.9% (83/185) and 44.3% (82/185), respectively. The median follow-up time was 1901 days (5.2 years), and the longest follow-up time was 3038 days (8.3 years). The OS, NRM, and relapse rates during the entire follow-up period were 63.8% (118/185), 14.1% (26/185), and 25.4% (47/185), respectively.

CMV infection combined with aGVHD is an independent risk factor for prognosis post-MST

It is clear that aGVHD and CMV infection contributes to inferior prognosis in HSCT. However, little is known regarding the prognostic impact of these factors on MST. To answer this question, Kendall’s tau-b correlation was first analyzed between these events. We found a significant positive association between aGVHD and CMV infection (Kendall’s tau-b=0.370, P < 0.001). Then NRM and OS were calculated in four groups of patients: CMV−aGVHD− (90/185), CMV+aGVHD− (46/185), CMV−aGVHD+ (12/185), and CMV+aGVHD+(37/185). The results showed that the cumulative incidence rate (CIR) of NRM was significantly higher (Fig. 1A) with concomitant lower OS (Fig. 1B) in the CMV+aGVHD+ double-positive (DP) group than in the other groups, which were defined as CMV−aGVHD− double-negative (DN) and CMV+aGVHD−/CMV−aGVHD+ single-positive (SP). Surprisingly, the CIR of NRM and OS were comparable among the patients in the DN and SP groups, suggesting that CMV infection and aGVHD increased NRM and decreased OS only when they occurred simultaneously in post-MST patients. The relapse rate was not affected by the co-occurrence of CMV infection and aGVHD (data not shown). The direct causes of death are summarized in Table 2, mainly infection and GVHD.

Since aGVHD and CMV infection affect the outcomes of transplantation when they occur simultaneously (CMV+aGVHD+), CMV+aGVHD+ was regarded as an independent factor in the following analysis. We performed a multivariate analysis of NRM and OS, and the results revealed two independent influencing factors: CMV+aGVHD+ and CD8+T cells reconstitution (Table 3).

Superior immune reconstitution protecting against the negative effects of aGVHD and CMV infection on transplantation outcomes

Given that CMV+aGVHD+ and CD8+T cells were both independent influential factors for NRM and OS, the effects of CD8+T cells on transplant outcomes in patients with CMV infection and aGVHD double positive were further evaluated. Based on our previous observations, patients were divided into three groups: (i) DN/SP, (ii) DP patients with CD8+ T IR, and (iii) DP patients without CD8+T IR. The cut-off value for IR was defined as the median number of CD8+T cells at day 90 (676 cells/µl). We compared the OS and NRM among the three subgroups. Our results showed that the DN/SP and DP with CD8+T IR groups had comparable NRM and OS rates, a survival rate that was more favorable than that of the DP without CD8+T IR group (Fig. 2A). Considering that CD19+B and CD4+T cells IR was correlated with NRM in univariate analysis, we also wanted to determine whether these two cell subsets affected the prognosis of DP patients. We found that patients who reached CD4+T cells IR also improved NMR and OS after CMV infection and aGVHD (Fig. 2B), but similar results were not found in CD19+B cells (data not shown). Furthermore, the immune reconstitution data from days 30 and 60 showed that CD8+T cell IR on day 60 had a protective effect on NRM (Fig. 2C). Taken together, these results suggest that superior IR has a protective effect against NRM and OS in co-occurring CMV infection and aGVHD.

CMV+aGVHD+ associated with delayed CD8+ T cell reconstitution

To evaluate the association between CMV+aGVHD+ and IR, IR in DP patients was compared to that in the DN/SP groups. Although the levels of CD19+B cells, CD4+T cells, and CD8+T cells were obviously delayed in univariate analysis (Table 4, Fig. 3A), multivariate analysis illustrated that reconstitution of CD8+T cells at day 90 was the event significantly correlated with co-occurring CMV infection and aGVHD (Table 4).

Recurrent CMV infections correlated with delayed CD8+T cell IR and adverse NRM

Early impaired T-cell reconstitution is a risk factor for CMV reactivation post-HSCT [3]; however, CMV infection is also related to boosted CMV-specific CD8+TEM (effector memory T) cell reconstitution [17], suggesting a complex network of associations between CMV infection and IR. Therefore, we speculated that a correlation exists between the frequency of CMV infection and IR. Patients with CMV infection were divided into three groups according to the frequency of CMV infection: infection once, recrudescence 2–4 times, and more than 5 times. In patients who experienced CMV infection more than 5 times, the reconstitution of CD3+T cells, especially CD8+T cells, but not CD4+T cells, was significantly lower than that in patients who underwent CMV infection less than five times on day 60 post-HSCT (Fig. 3B), and the increased frequency of CMV infection increased the risk of NRM (Fig. 3C).

Discussion

Patients with both aGVHD and CMV infection who underwent HLA-matched sibling transplantation had significantly higher non-relapse mortality (NRM), lower overall survival (OS), and delayed IR. Multivariate analyses showed that aGVHD combined with CMV infection, and delayed CD8+T cells IR were independent risk factors for prognosis post-MST. Recurrent CMV infections are associated with inferior CD8+T cells reconstitution. Optimal T-cell reconstitution ameliorated the adverse outcomes of CMV infection and aGVHD.

CMV infection and aGVHD, the most common complications after transplantation, are associated with poor prognosis. Our study considered a strong correlation between aGVHD occurrence and CMV infection, and patients were divided into four groups: CMV−aGVHD−, CMV+aGVHD−, CMV-aGVHD+, and CMV+aGVHD+. The results showed that NRM and OS were not affected by CMV infection and aGVHD occurrence alone; however, NRM and OS were significantly affected only when CMV infection and aGVHD occurred simultaneously. Compared with other studies that showed that CMV infection or aGVHD alone could affect transplant outcomes, our study enrolled patients who underwent HLA-matched sibling transplantation. The incidence of viral infections was lower in the patients who underwent HLA-matched transplantation [18]. The incidence of aGVHD was also lower in patients who underwent HLA-matched related transplantation than in those who underwent haploidentical-related, matched-unrelated, or mismatched-unrelated transplantation [19]. Meanwhile, the GVL effect of HLA-matched transplantation was weaker than that of haploidentical transplantation [20, 21]; therefore, our results showed that NRM decreased and OS increased only when CMV infection and aGVHD occurred simultaneously in MST patients.

Poor IR can lead to an increased risk of infection, relapse of the primary disease, and secondary malignancies, therefore, IR plays a crucial role in HSCT success. In our study, the results revealed that aGVHD combined with CMV infection was highly associated with delayed IR, recurrent CMV infections were associated with delayed CD8+T cells IR, and aGVHD was positively correlated with CMV infection, indicating that CMV infection, aGVHD occurrence, and IR can influence each other directly and indirectly, which is consistent with previous studies [3, 4, 12–14]. aGVHD can inhibit post-transplant IR and thus provide a more favorable environment for CMV reactivation, leading to more serious outcomes. On the one hand, steroids as the first-line therapy for aGVHD, the dose of steroids and the cumulative amount of steroids in peripheral blood are significantly correlated with impaired CMV-specific CD4+T and CD8+T functional reestablishment in a dose-dependent manner [22]. On the other hand, aGVHD could directly affect IR, aGVHD has been shown to affect the BM niche and IR post-HSCT [4], and an animal study confirmed that aGVHD can induce severe dendritic cell defects, resulting in the inability to produce CMV-specific CD8+ T cells, which greatly limits the antiviral response [12]. Other studies have revealed that delayed IR, including T and NK cells, can increase the risk of CMV infection [3, 9], and CMV infection also leads to IR dysregulation through the massive expansion of CMV-specific CD8+TEM cells, resulting in a linked contraction of other T cell subsets [17].

Our data showed that the number of CD8+ T cells on day 60 was associated with recurrent CMV infections. The number of CD4+ and CD8+ T cells was positively correlated with the number and function of CMV-specific CD4+ and CD8+ T cells, and the number of CD8+ T cells was not only correlated with the number and function of CMV-specific CD8+ T cells but also with the function of CMV-specific CD4+T cell [22]. Therefore, impaired CD8+ T-cell reconstruction on day 60 results in an impaired response of CMV-specific CD4+ and CD8+ T cells and a weakened ability to eliminate viruses, leading to recurrent CMV infection and a poor prognosis. Our data also showed that superior immune reconstruction can protect NRM and OS after CMV infection and aGVHD.

The median value was used to define the threshold of cellular IR. In other studies, the threshold for CD4+ T and CD8+ T IR or lymphocytopenia was usually 50 cells/µl [7, 23–27]; In our study, the CD4+ T cell count was >50 cells/µl in 89% (139/155) of the patients, whereas that for CD8+ T cells was >50 cells/µl in 85% (132/155) of the patients, even on day 30 post-transplantation; hence, the estimated value in previous studies did not coincide with the data in our study. Several differences were observed between our transplantation conditions and those in other studies: (1) the target population comprised mostly pediatric patients; (2) the primary sources of grafts included umbilical cord blood, bone marrow, and peripheral blood; (3) transplant donors included HLA-matched related donors, haploidentical-related donors, matched-unrelated donors, and mismatched-unrelated donors; (4) grafts with T-cell depletion; and (5) ATG was used as the conditioning regimen. In our cohort, (1) the target population was mainly adult patients (173/185) with a median age of 41 years; (2) the main sources of grafts were bone marrow and peripheral blood, rather than umbilical cord blood; (3) all patients underwent HLA-matched sibling transplantation; (4) grafts without T-cell depletion; and (5) ATG was not used in conditioning. Compared with bone marrow and peripheral blood stem cell transplantation, umbilical cord blood transplantation has delayed IR and poor virus control capability [28]. HLA-matched related recipients have better IR than other types of transplantation donors [21], and the use of ATG can significantly affect IR post-transplant [23, 24, 27]. Therefore, different IR thresholds should be defined according to IR values under distinct transplant conditions.

Since superior IR can protect NRM and OS after aGVHD and CMV infection, and CMV infection generally occurs later than aGVHD, the median times of CMV infection and aGVHD were 49 and 33 days post-HCT, respectively, in our cohort. Therefore, IR recovery can be promoted at an early stage of CMV infection to improve transplantation outcomes, and infusion of CMV-specific T cells after CMV infection has been reported to improve outcomes [29].

Our study has some limitations. (1) This was a retrospective study; the duration of immune surveillance was one month. Hence, further details regarding IR time cannot be obtained; (2) IR assays are less robust, other immune cells, such as NK cells, may be involved; and (3) only HLA-matched sibling transplantation was performed in this study. Therefore, the data may not be applicable to other transplantation types.

In conclusion, the co-occurrence of aGVHD and CMV infection was associated with poor CD8+ T-cell IR and inferior post-MST outcomes, and there was a strong correlation between aGVHD occurrence and CMV infection. However, preferable IR could protect against the negative effects of aGVHD and CMV infection on the transplant outcomes. Therefore, further treatment can be performed early after CMV infection and aGVHD based on the amount of cell IR, to improve the transplant outcomes.

Abbreviations

Abbreviations
 
• aGVHD

  acute graft-verse-host disease

 
• Allo

  allogeneic

 
• CMV

  Cytomegalovirus

 
• HSCT

  Hematopoietic Stem Cell Transplantation

 
• MST

  HLA-matched transplantation

 
• NRM

  non-relapse mortality

 
• OS

  overall survival

 
• IR

  immune reconstitution.

Acknowledgments

The authors thank all of the core facilities at the Peking University Institute of Hematology for sample collection. We would like to thank Editage (www.editage.cn) for English language editing.

Funding

This study was supported by grants from the Beijing Municipal Science and Technology Commission (no. Z171100001017098), National Natural Science Foundation of China (Grant Nos. 81670166, 81870140, 82070184, and 81370666), Peking University People’s Hospital Research and Development Funds (No. RDX2019-14; RDL2021-01).

Conflicts of interest

The authors declare no financial or commercial conflict of interest.

Author contributions

ZXY was responsible for conceiving and designing the study; ZXS, CYJ, LM, MXD, SYQ, ZYY, WY, XLP, ZXH, LKY, and HXJ performed the clinical examination; ZW was responsible for supplementing case information; FZY and HTT contributed to analyzing and interpreting the data and writing the manuscript; and all authors read and approved the final manuscript.

Ethical approval

This study was approved by the Human Ethical Systems Review Committee of Peking University People’s Hospital.

Patient consent

All patients provided written informed consent before their entry into the study in accordance with the Declaration of Helsinki.

Data availability

The data presented in this manuscript are included in the paper.

References

Author notes