The establishment of a cytomegalovirus -specific CD8+ T-cell threshold by kinetic modeling for the prediction of post-hemopoietic stem cell transplant reactivation

Summary The dynamic interaction between the CMV virus and host immune response remains obscure, thus hindering the diagnosis and therapeutic management of patients with HSCT. The current diagnosis of CMV viremia depends on viral load estimation. Medical intervention based on viral load, can be unnecessary or poorly timed for many patients. Here we examined the clinical features and blood samples of patients with HSCT and assessed the CMV reactivation kinetics and corresponding CMV antigen-specific T-cell response in individual patients based on a peptide pool stimulation T-cell assay, which showed that CMV-specific CD8+ T cells were more suitable to be a diagnosis indicator for suppressing CMV reactivation. Using ROC analysis, we defined and verified a CMV-specific CD8+ T-cell counts threshold (925 cells/106 PBMCs) as an indicator of CMV reactivation post-HSCT, and suggested that use of this threshold would provide more accurate guidance for prompt medication and better management of CMV infection post-HSCT.


INTRODUCTION
Cytomegalovirus (CMV) infection is one of the major causes of morbidity and mortality following allogeneic hemopoietic stem cell transplant (HSCT), which is used to treat a variety of blood disorders, including non-malignant diseases, e.g., aplastic anemia, and hematologic malignancy, e.g., leukemia, lymphoma and multiple myeloma (de Koning et al., 2016;Simons et al., 2019). For a period of time after HSCT, recipients' immunity remains depleted and suppressed while the reconstitution of donorderived host immunity is in progress, leaving HSCT recipients vulnerable to opportunistic infections. Indeed, at this stage, pre-existing latent CMV from the host or graft can flare up within the recipient (Liu et al., 2015;Moss and Rickinson, 2005). It is known that CMV reactivation occurs in approximately 60% to 70% of seropositive patients with HSCT within the first 100 days post-HSCT, and without prompt additional viral suppression treatment up to 10% of patients progress into potentially fatal CMV diseases which include pneumonitis, gastritis, enteritis, colitis, hepatitis, retinitis, and encephalitis (Boeckh et al., 2003;Einsele et al., 2020;Goldsmith et al., 2016;Hodowanec et al., 2020;Lee et al., 2017;Maertens and Lyon, 2017).
Currently, CMV reactivation is diagnosed through the estimation of blood CMV titer by CMV-DNA and -antigen (pp65) tests (Berth et al., 2016;Kamei et al., 2016). However, these snapshots of blood viral load are far from satisfactory for the clinical assessment of overall disease severity or prediction of disease outcomes. A CMV-DNA positive result does not always manifest any CMV disease, whereas a negative CMV-DNA result cannot rule out the development of CMV diseases. Consequently, in the hospitals of our study cohort, a 3-month prophylactic antiviral therapy with antiviral agents (ganciclovir, valganciclovir or foscarnet) is routinely applied following HSCT to reduce the chance of CMV recurrence (Boeckh and Ljungman, 2009;Sellar and Peggs, 2012). Then, pre-emptive approach after the first 90 days is performed involving close viral load monitoring and antiviral treatment for significant viral reactivation. However, the prolonged use of these antiviral drugs can cause adverse effects on bone marrow and kidneys and may lead to the selection of drug-resistant strains of CMV (Boeckh and Ljungman, 2009;Heagy et al., 1991;Ljungman et al., 2010). Moreover, the high costs of anti-viral therapy impose a huge financial burden on patients whose medical costs are not fully covered by insurance. Thus, from a clinical perspective, there is an urgent need to develop improved diagnostic methods to monitor CMV reactivation and guide targeted antiviral therapy.
Previous studies, in a mouse model and in human HSCT cases, have shown that CD8 + T-cells, CD4 + T-cells, natural killer (NK) cells, and B cells all play important roles in controlling CMV reactivation (Alegre, 2019;Apiwattanakul et al., 2020;Chang et al., 2014;Rogers et al., 2020). However, the reconstitution of recipients' adaptive immunity through the engraftment of donor stem cells is a slow process, which occurs over months to years (van den Brink et al., 2015). In addition, the re-establishment of host immunity can be affected by multiple HSCT-related factors such as recipient and donor CMV serostatus, the stem cell source, the degree of human-leukocyte-antigen (HLA) matching, infections, presence of graft-versus-host disease (GVHD) and degree of corticosteroids use for immune suppression (Pei et al., 2017). The variations of these factors in individual patients make it difficult to accurately evaluate the true extent of immune reconstitution after HSCT. To date, no standardized index or threshold has been established to indicate an effective immunity for controlling CMV reactivation (Camargo et al., 2019;Tey et al., 2013). Furthermore, during the immune reconstitution, fluctuations of recipients' immunity occur frequently, which may allow CMV recurrence. Therefore, a better understanding of the dynamic relationship between host immune responses, particularly T-cell immunity, and CMV reactivation is important for better management of post-HSCT CMV-mediated complications (Wagner-Drouet et al., 2021).
In this study, we analyzed blood samples of 88 patients from Guangzhou First People's Hospital (GFPH) and 43 patients from the First Affiliated Hospital of Guangzhou Medical University (GMUH) who had undergone HSCT. CMV-specific T-cell immunity (CD4 + and CD8 + ) assays were performed based on a peptide pool stimulation T-cell assay and CMV titers were measured (via DNA) at various timepoints over a period of 90 days to 3 years post-HSCT. Reiterative computational data-fitting allowed us to develop a mathematical model through which a significant correlation was established between CMV reactivation kinetics and the antigen-specific CD8 + T-cell response. Importantly, by analysis of patients from two different hospitals, we were able to define a threshold of CMV-specific CD8 + T-cell counts (per million PBMCs) that corresponded, in most cases, to CMV containment. We then verified the use and robustness of the threshold in separate patient cohorts. Thus, we defined a new parameter: CMV-specific CD8 + T-cell counts per million PBMCs, which has the potential to be used as an effective cue for anti-viral therapy.

Clinical characteristics of patients and incidence of cytomegalovirus viremia following hemopoietic stem cell transplant
In this study, two cohorts of patients with HSCT, diagnosed with acquired severe aplastic anemia (SAA) (cohort I: 64 patients) and hematologic malignancy (HM) (cohort II: 24 patients), respectively, were recruited from Guangzhou First People's Hospital (GFPH), the largest SAA diagnosis and treatment center in South China (Table 1). Additionally, 21 patients with HSCT, each diagnosed with various forms of HM, were recruited from a different hospital, the First Affiliated Hospital of Guangzhou Medical University (GMUH) (Cohort III, Table 1). The clinical characteristics of the patients, including donor source, stem cell source, donor/recipient CMV serostatus, and the presence or absence of GVHD, were summarized in Table 1.
To analyze CMV-DNAemia occurrence in the three cohorts, we performed quantitative real-time PCR using plasma collected at various timepoints between 2 weeks before and 3 years after HSCT. The diagnostic criterion of CMV viremia was defined as 500 copies/mL in plasma (clinical threshold for treating). Based on the medical records and laboratory data, the occurrence of viremia over time was assessed. For the patient cohorts from GFPH, the accumulative CMV incidences were 80.3% (74.7% at d60) and 61.4% (47.8% at d60) for HSCT patients with SAA (cohort I) and HM (cohort II), respectively (Figures S1A and S1B). For cohort III (patients with HM from GMUH), the overall cumulative CMV incidence was 61.7% (51.3% at d60) ( Figure S1C), similar to that of cohort II (p = 0.8443). Collectively, these results show that the incidence of CMV reactivation rises rapidly before d60 and reaches a plateau at around d100, in agreement with previous clinical observations (Green et al., 2012(Green et al., , 2013 The large set of PCR data (1775 samples from 88 patients) obtained from the GFPH cohorts (I & II) allowed us to investigate the kinetics of CMV reactivation. We first produced a heat map for both patients with SAA and HM that displays the distribution and level of CMV viremia over a period of 330 days ( Figure 1A). A very low CMV incidence was seen within the first 30 days after HSCT ( Figures 1A and 1B). This was followed by a large increase in the detection of CMV between d30-60. After d60, the incidence of CMV detection declined ( Figures 1A and 1B).
To further analyze the dynamic distribution of CMV viral loads, we selected patients who had one episode of CMV reactivation within 90 days post-HSCT (43 from the 88 patients) ( Figure 1C) and fitted their DNA data into a single ordinary differential equation (for details see STAR methods). After reiterate computational modeling, a representative CMV-DNA kinetic curve was generated ( Figure 1D) from the data of individual cases ( Figure S2). Consistent with our conclusions from the heat map visualization, the bulk of CMV iScience Article reactivation occurred between d29-64 ( Figure 1E). In addition, we also obtained the following important information with respect to post-HSCT CMV reactivation: (i) the median latency period for CMV replication to reach the levels of >500 copies/mL in plasma was 25 days with a minimum of 17 days and a maximum of 35 days (min-max range: 17-35 days); (ii) the median period for CMV to reach the peak level (2403.4 iScience Article copies/mL) was 44 days (min-max range: 29-64 days); and (iii) the median duration of infection resulting from the first episode of CMV reactivation was 51 days (min-max range: 33-63 days) ( Figure 1E).

The dynamics of cytomegalovirus-specific CD8 + T-cell responses after hemopoietic stem cell transplant
To understand the dynamics of post-HSCT T-cell immune reconstitution, based on a peptide pool stimulation T-cell assay, we measured the total CMV-specific (IFNg + ) CD8 + T-cells from the blood samples of the GFPH cohorts (88 patients) collected at 280 time points over 330 days and generated a heat map (Figures 2A and S3). A likelihood ratio test of the experimental data revealed that the levels of CMV-specific CD8 + T-cells produced within a window of 35 days between d50-85 were significantly higher than those observed between d15-50 (p < 0.001, n = 56) but lower than those seen between d85-120 (p = 0.0068, n = 53) ( Figure 2B).
Next, we generated a CMV-specific CD8 + T-cell response curve by fitting a single ordinary differential equation with the data from the 68 (out of 88) patients ( Figures 2C and S4) who had at least one CD8 + T-cell measurement within 180 days post-HSCT (as described in STAR methods). The response curve (Figure 2D) displayed a temporal pattern of three phases (I, II, and III) which corresponded to the three post-HSCT CMV infection phases (phase I: latency, phase II: duration of reactivation, and phase III: resolution). During phase I, the titers of the CMV-specific CD8 + T-cells in most patients remained extremely low (9.1 per million PBMCs) and the median length of this phase was 20 days (range: 10-44 days) ( Figure 2E). Given that the latency period for CMV viremia was 25 days (range: 17-35 days) ( Figure 1E), we inferred that some CMV antigens were expressed and could stimulate T-cell response, which could be detected by our T-cell assay before CMV-DNA reached 500 copies/mL to be defined as CMV viremia, our data suggested the CD8 + T-cell response commenced during or immediately after the start of CMV reactivation.
In phase II, the CMV-specific CD8 + T-cells increased rapidly, eventually reaching the median level of 1055 cells/million PBMCs prior to the viral resolution phase ( Figures 2D and 2E). During phase III, the levels of the CMV-specific CD8 + T-cells began to plateau with a median of 1976.6 CMV-specific CD8 + T-cells/million PBMCs ( Figures 2D and 2E) around d180.
We also examined the dynamic changes of CMV-specific CD8 + T cells for 25 patients with no detectable CMV viremia in the blood (other patients were diagnosed with single CMV viremia (n = 50) or repeated CMV viremia (n = 13)). Data showed that over time there was a significant increase in the levels of CMV-specific CD8 + T-cells in these 25 patients ( Figures S5A and S5B). It's probably owing to the fact that the viral load in plasma didn't meet the diagnostic (<500 copies/mL), or CMV reactivation might have occurred in tissues other than blood. Furthermore, we couldn't exclude the possibility that CMV-specific CD8 + T-cells were recovered by other immune mechanisms thus preventing CMV reactivation.

The dynamics of cytomegalovirus-specific CD4 + T-cell responses after hemopoietic stem cell transplant
To analyze the dynamics of CMV-specific CD4 + T-cell responses post-HSCT, we used the same blood samples of the GFPH cohorts (I and II) for the analysis of the CD8 + T-cell responses to generate a CD4 + T-cell response heatmap ( Figure 3A) and perform a likelihood ratio test of CMV-specific CD4 + T-cell levels over three different periods ( Figure 3B). Unlike what was observed for CD8 + T-cells, there was no significant difference in the CD4 + levels between (d15-50], (d50-85] and (d85-d120] ( Figure 3B). As in our above analysis of CD8 + T-cells, 68 (out of 88) patients, who had at least one CD4 + T-cell measurement within 180 days post-HSCT, were selected in the fitting of the CD4 + T-cell model ( Figure 3C). The frequency of responding CD4 + T-cells producing IFN-g was around 396.3 (309.2-422.6) cells/million PBMCs upon viral resolution, which was much lower than for CD8 + T-cells. At d180, the mean level of virus-specific CD4 + slightly decreased to 158.6 (2.54-788.5) cells/million PBMCs ( Figures 3D and 3E), approximately 1/10 of those of the CD8 + T-cells ( Figures 2E and 3E), showing that although CMV reactivation was able to elicit a CD4 + T-cell response , it increased during CMV viremia then declined upon suppression of the virus. The individual patient data fitting of the CD4 + T-cell model was shown in Figure S6. The lower magnitude and decline after the viral resolution of CMV-specific CD4 + T-cells, compared to CD8 + T-cells, were further exemplified by four representative patients with multiple time points across 180 days where CD8 + or CD4 + T-cell counts and data fitting curves were compared ( Figures S7A-S7C).

OPEN ACCESS
iScience 25, 105340, November 18, 2022 5 iScience Article Defining an antigen-specific-CD8 + T-cell threshold as an indicator for cytomegalovirus containment The demonstration of a strong correlation of the antigen-specific-CD8 + T-cell counts with the suppression of CMV replication and a close connection of the kinetic profiles of CMV distribution and CD8 + response ( Figure 2D) suggested that antigen-specific CD8 + T-cells could be used as an indicator for CMV containment. Hence, we next sought to define a CMV-specific CD8 + T-cell resolution threshold for the prediction of CMV reactivation.  Tables S2-S5), with corresponding CMV-DNA and CMV-specific CD8 + T-cell data, were used in a receiver operating characteristic curve (ROC) analysis to evaluate the performance of the immunological detection of CMV viremia (Figure 4). We plotted the true positive rate (sensitivity) against false-positive rate (represented as: 1-specificity). The area under the curve (AUC) calculated using the trapezoid rule (see STAR methods) was 0.727 ( Figure 4A), indicating an effective measure of accuracy in using immunological detection as a diagnostic test (Hajian-Tilaki, 2013). Since this study aims to achieve a safe threshold to guide the start and end of anti-viral treatment, sensitivity is more important than specificity, therefore, we chose to concentrate on a part of the ROC curve in which the sensitivity rose sharply (0.74-0.88) without much change in specificity (0.52-0.54) ( Figure 4A, indicated in the  Table S1). At this threshold, 62.8% had expected diagnostic outcomes, which included 96 T + V-timpoints (CD8 + T-cells (T value) above 925 cells/10 6 PBMCs and CMV loads (V value) below 500 copies/mL) and 59 T-V+ timepoints (CD8 + T-cells below 925 cells/10 6 PBMCs and CMV-DNA levels above 500 copies/mL) ( Figure 4C and Tables S1-S3). In contrast, 12 of 247 timepoints showed CMV virus titers higher than 500 copies/mL with corresponding CMV-specific CD8 + T-cell counts above the T-threshold (T + V+) and 80 timepoints showed no CMV viremia detected while CMV + CD8 + T-cells were lower than the T-threshold (T-V-) ( Figure 4C and Tables S1, S4, and S5).
Based on the dynamic computational model ( Figure 2D), the time to reach the threshold (925 specific CD8 + T-cells/million PBMCs) was predicted to be 69 days (range: 35-112 days). Given that the presence of patients with CMV viremia following HSCT persists between d17 (the start of latency) and d98 (the sum of the longest latency period, 35 days, and the maximum duration of infection, 63 days, Figure 1E), the clearance of viral replication correlated well to the magnitude of CMV-specific CD8 + T-cell response. Furthermore, our model also indicated that the median level of CMV-specific CD8 + T-cell counts at the initial point of the viral resolution phase was 1055 (range: 12-2913 cells/million PBMCs) ( Figures 2D and 2E).

Threshold were verified in the clinical context and showed great power in predicting the status of cytomegalovirus reactivation
To evaluate the power of T-threshold in predicting CMV reactivation clearance, we studied the kinetics of CMV-DNA and CMV-specific CD8 + T-cell from multiple timepoints ( Figure 5 and Tables S2-S5). Interestingly, based on a total of 155 timepoints with T+V-and T-V+, we found the CD8 + T-cells were maintained above the T-threshold, at the same time, the CMV replication was effectively suppressed, in contrast, when CD8 + T were lower than threshold, CMV-DNA were detected ( Figure 5A and Tables S2 and S3). It showed that the defined T-threshold value reflects the switch point between CMV reactivation and CMV-specific CD8 + T immunity within individual patients with HSCT ( Figure 5D).
Case analyses of the 12T+V+ timepoints which comprised 4.9% of patients showed that, without the complication of a relapse of pre-HSCT disease, when patients' T-cell counts climbed above the T-threshold of 925 cells/10 6 PBMCs, their blood CMV would be cleared with a short delay of 2-9 days (Figure 5B and Table S4). Thus, the T-threshold could predict subsequent viral clearance and could be used as a safe indication index for stopping anti-viral therapy ( Figure 5E).  (Table S5). Of them, 20 timepoints from 6 patients were found to fail to generate adequate levels of CMV-specific CD8 + T-cells over a prolonged period after HSCT and suffered multiple episodes of CMV viremia ( Figure 5C, blue dot in green cycle). Consequently, four of them developed CMV retinitis, above facts showed only after the establishment of adequate levels of T-cell immunity would be able to avoid further CMV infection, emphasizing the importance of using the immunological test combining CMV-DNA test in monitoring the development of post-HSCT CMV reactivation. Thus we suggested that patients with no detectable CMV viremia in the blood but with below-threshold counts of CMV-specific CD8 + T-cells still require antiviral treatment ( Figures 5F and 6B). For another 57 timepoints in 29 patients, T-cell immunity levels were still recovering or fluctuating below the threshold but CMV-DNA was not detected, and it occurred mostly within the first 100 days following HSCT ( Figure 5C, blue dot in orange cycle). Under such circumstances, as almost all of these T-V-timepoints were seen within the period of compulsory prophylactic antiviral therapy, they would not affect post-HSCT CMV management ( Figures 5F  and 6B).
In sum, combining the CMV-DNA test, our threshold showed strong power in predicting the status of CMV reactivation and guiding for when to start and stop the anti-viral therapy.

DISCUSSION
Cellular immunity has long been known to play an important role in the control of CMV infection (Klenerman and Oxenius, 2016). However, to date, the dynamic interplay between CMV infection and host T-cell immunity is poorly understood. In this study, we examined the clinical characteristics of patients with HSCT from two separate medical settings and analyzed the kinetic interactions between CMV reactivation and the CMV-specific T-cell response. Owing to the very high CMV-infection rate in the Chinese population, a universal 90-day prophylactic therapy with antiviral drugs is given to all patients with HSCT. Under these conditions, the CMV viremia curve generated through analysis of blood samples of patients at various timepoints displays a typical distribution with the majority of CMV incidence occurring between d17 and d98 after HSCT and the peak of Although lower than the CD8 + T-cell response, the synchrony of the kinetic curves of CD4 + T-cells and CMV-DNA suggests that CD4 + T-cells may also contribute to the inhibition of CMV replication. However, in contrast to CD8 + T-cells, which remained high after 180 days, CD4 + T-cell counts in a significant number of patients declined after their CMV viremia was suppressed. The different patterns of CD4 + and CD8 + T-cell responses may reflect the separate pathways used for the surveillance of CMV and presentation of viral antigens. It is known that CD4 + T-cells predominantly recognize CMV structural proteins whereas CD8 + T-cells can recognize non-structural proteins encoded by immediate-early gene loci, inhibiting CMV transcriptional reactivation before the formation of viral particles (Simon et al., 2006). In addition, the differential responses of CD8 + and CD4 + T-cells to post-HSCT CMV reactivation are in agreement with the view that CMV-specific CD8 + T cells are more suitable to be a diagnosis indicator for suppressing CMV reactivation whereas CD4 + T-cells are important for the maintenance of antiviral immunity including expansion and persistence of CD8 + T-cells (Boeckh and Ljungman, 2009;Meesing and Razonable, 2018).
The ultimate application of using CMV-specific CD8 + T-cell counts to the clinical prognostication of post-HSCT CMV reactivation relies on the standardization of quantification and methodologies for the evaluation of this biomarker. To this end, we used the flow cytometry intracellular cytokine staining (ICS) method to process 247 HSCT samples, then defined the CD8 + T-cell counts, R925 cells/10 6 PBMCs, as the resolution threshold for CMV reactivation using ROC analysis. Recently, Messing and Razonable have reported the correlation of peripheral blood absolute lymphocyte count (PBALC) with CMV infection and proposed the PBALC value, %830/mL, as a clinically relevant threshold for the prediction of CMV infection. In addition, two other studies have demonstrated the utility of the simple and high throughput QuantiFERON-CMV assay to monitor CMV-specific CD8 + T-cells for the prediction of CMV infection in patients with HSCT (Tey et al., 2013;Yong et al., 2017). However, no cut-off points for CD8 + T-cell quantification were defined in these studies. Moreover, in a previous study, Angeles Clari et al. compared the performance of QuantiFERON-CMV assay with that of the ICS method and found the latter to be more sensitive in the detection of CMV-specific CD8 + T-cells for patients with HSCT (Angeles Clari et al., 2012). Our case analyses showed that CMV-specific CD8 + T-cell counts were particularly useful for assessing secondary and recurrence CMV reactivation after the prophylactic period (the first 90 days post-HSCT) and that a stable reconstitution of CMV-specific T-cell immunity as measured by the continuous above-threshold levels of CD8 + T-cells ensured the suppression and clearance of CMV within patients with HSCT. Even with occasional rises in viral replication, the CMV titers would decline to an undetectable level in around 7 days without the administration of antiviral drugs. On the other hand, the immune reconstitution of patients with HSCT can easily be hampered by anti-GVHD treatments or relapse of leukemia, resulting in CD8 + T-cell levels below the threshold for a prolonged period. Under these circumstances, a further pre-emptive antiviral therapy is recommended to prevent CMV-associated diseases.
Patients developing CMV disease correlating with low CD8 + T-cell counts, but without detectable CMV-DNA in plasma, suggesting that CMV reactivation might have occurred in distal tissues/organs and have not spilled over to blood with detectable quantity, supporting our hypothesis that the inability for the CD8 + T-cells to reach the resolution threshold of 925 cells/10 6 PBMCs serves as a warning sign for CMV reactivation even in the absence of detectable levels of CMV in the plasma.
In fact, if we use CD8 + T cells measured after the time of prophylaxis (90 days post-HSCT) to predict viremia that comes at the same time, the sensitivity and specificity of the virological test will be 44.7% and 91.2%, which means that the rate of missed diagnosis (mistakenly to recommend withdrawal) and misdiagnosis (over-medication) of CMV-DNA test is 55.3% and 8.8% (Table S6). Traditionally, after prophylaxis treatment within 90 days post-HSCT, only the CMV-DNA test is used to monitor CMV reactivation, if CMV-DNA positive (V+), antiviral treatment continues, otherwise it stops ( Figure 6A). For future CMV antiviral therapy after 90 days prophylaxis treatment, combining T-cell assay and CMV-DNA test, we want to address two exceptional circumstances ( Figure 6B): 1) when CMV-DNA levels above 500 copies/mL (V+) and T-cell counts higher than T-threshold (T+) are detected after 90 days post-HSCT, the antiviral treatment is advised to stop. 2) when V-and T-are detected after 90 days post-HSCT, we strongly suggested that the antiviral therapy must be implemented although traditionally V-is considering not risky.
HSCT recipients are given prophylaxis or are closely preemptively monitored for the first 100 days after transplant in many countries (Einsele et al., 2020). Traditionally, the pre-emptive approach involves close viral load monitoring and treatment for significant viral reactivation ( Figure 6A). The problem that this type of data could be applied to solving is that it becomes difficult to continue monitoring patients weekly for CMV viral load blood draws indefinitely after transplant. Thus, having a T-cell marker measured around day 90 or 100 that could predict later viremia would be quite helpful. We could use such a marker to avoid having to measure additional viral loads in patients with protective responses after the critical reactivation period.

Conclusions
The dynamic interaction between CMV virus and host immune response remains obscure, thus hindering the diagnosis and therapeutic management of patients with HSCT. In this work, we assessed the CMV reactivation kinetics and corresponding CMV antigen-specific T-cell response in individual patients, which showed that compared to CMV-specific CD4 + T-cells, CMV-specific CD8 + T-cells are more suitable to be a diagnostic indicator for suppressing CMV reactivation. Then, our case analyses indicated that CMVspecific CD8 + T-cell counts are particularly useful for assessing CMV recurrence after the prophylactic period (the first 90 days post-HSCT) and that a stable reconstitution of CMV-specific T-cell immunity as measured by the continuous above-threshold levels of CD8 + T-cells ensured the suppression and clearance of CMV within patients with HSCT. Based on ROC analysis, we defined a CMV-specific CD8 + T-cell threshold (925 cells/10 6 PBMCs) for the immunological detection of CMV viremia. We addressed the utility of the T-cell threshold in two exceptional circumstances: 1) when CMV-DNA levels above 500 copies/mL (V+) and T-cell counts higher than T-threshold (T+) were detected after 90 days post-HSCT, the antiviral treatment was advised to stop. 2) when V-and T-were detected after 90 days post-HSCT, we strongly suggested that the antiviral therapy must be implemented although traditionally V-is considering not risky.
Combining CMV-DNA test, our data suggest that the use of this threshold would provide more accurate guidance for prompt medication and better management of CMV infection post-HSCT. iScience Article CMV reactivation, there are limitations in this study. Owing to around 95% high prevalence of CMV infection in China, we could not validate the threshold those D-/R+ (high-risk) or even D+/R-(low-risk) patients. As a retrospective study, inconsistent timepoints over a large period of time were collected, which had to use complicated fitting mathematical model for each patient for clear interpretation, and our modeling and kinetics also could not tell the causal relationship between CD8 + T-cells and CMV reactivation. Most importantly, we need to point out our T-cell assay should combine CMV-DNA test to guide only after 90 days post-HSCT, instead of a surrogate assay for the CMV-DNA test. Furthermore, this is a bi-centre investigation from one geological area. Scale-up studies with more participants from multiple clinical settings and different geological locations will improve the statistical significance of this analysis and strengthen the conclusion from our investigation.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following: