Clinical Factors Influencing Phenotype of HCMV-Specific CD8+ T Cells and HCMV-Induced Interferon-Gamma Production after Allogeneic Stem Cells Transplantation

Human cytomegalovirus (HCMV) infection causes significant morbidity and mortality after hematopoietic stem cell transplantation (HSCT). In this work, we characterized the phenotype and interferon-gamma (INF-γ) production of HCMV-specific T cells using QuantiFERON-HCMV assay in 26 patients 6 months after HSCT. We analysed whether these two parameters were associated with clinical variables. Our results showed that the patients receiving stem cells from donors ≥40 years old were 12 times more likely to have HCMV-specific CD8+ T cells with “differentiated phenotype” (CD45RA+CCR7+ ≤6.7% and CD28+ ≤30%) than patients grafted from donors <40 years old (OR = 12; P = 0.014). In addition, a detectable IFN-γ production in response to HCMV peptides (cutoff 0.2 IU/mL IFN-γ; “reactive” QuantiFERON-HCMV test) was statistically associated with HCMV replication after transplantation (OR = 11; P = 0.026), recipients ≥40 versus <40 years old (OR = 11; P = 0.026), and the use of peripheral blood versus bone marrow as stem cell source (OR = 17.5; P = 0.024). In conclusion, donor age is the only factor significantly associated with the presence of the “differentiated phenotype” in HCMV-specific CD8+ T cells, whereas HCMV replication after transplantation, recipient age, and stem cell source are the factors associated with the production of IFN-γ in response to HCMV epitopes.


Introduction
Human cytomegalovirus (HCMV) infection is a major cause of morbidity and mortality in subjects who undergo allogeneic stem cell transplantation (HSCT) due to the long period of immunode�ciency aer SCT [1][2][3]. HCMV-speci�c immune reconstitution aer HSCT plays a critical role in preventing HCMV infection and disease. Lack of this T-cell HCMV-speci�c subpopulation is associated with a higher risk of HCMV infection, as has been reported in HCMV-seropositive patients receiving an HSCT from HCMV-seronegative donors [4][5][6][7][8]. e magnitude of HCMV-speci�c CD8+ T-cell recovery predicts the risk of progressive HCMV infection [8,9], but HCMV replication aer HSCT also depends on the presence of dysfunctional HCMV-speci�c CD8+ T cells rather than on the absolute numbers of HCMV-speci�c T cells [10,11].
Aer encountering HCMV, naive T cells proliferate and become effector memory HCMV-speci�c CD8+ T cells, which exert an effector function in peripheral tissues and exhibit a differentiated phenotype. During this process, the downregulation of some costimulatory surface molecules (such as CD28 or CD27) and an increase in interferongamma (IFN-) production have been reported [12][13][14][15]. erefore, a decrease in the percentage of naive T cells and a parallel increase in the percentage of differentiated effector memory T cells (mostly CD28−) are observed aer HCMV infection. In addition, these phenotypic changes on HCMV-speci�c CD8+ T cells have also been linked with immunosenescence, suggesting that HCMV accelerates the process of age-associated immune function impairment [16][17][18][19]. In the HSCT setting, a correlation between the absolute number of circulating CD8+ HCMV-speci�c T cells in the recipient and the risk of HCMV infection has been demonstrated [8]. In addition, the number of less-differentiated CD8+ HCMV-speci�c T cells in the donor is associated with protection of HCMV infection. ese donor lymphocytes acquire a differentiated pro�le and restricted function in the recipient aer HSCT [10,11]. erefore, assessment of the maturation status and functional capability of circulating HCMV-speci�c T lymphocytes aer HSCT and the impact of clinical factors in the HCMV-speci�c immune recovery is crucial to improving our management of HCMV infection in these patients.
In this work, we have studied IFN-production in response to HCMV peptides and the phenotype of HCMV-speci�c CD8+ T cells in a group of HSCT patients 6 months aer allogeneic transplantation. In this cross-sectional study, we analyse whether these two parameters are associated with HCMV replication aer transplantation as well as other clinical variables such as donor and recipient age, donor and recipient serostatus, and stem cell source. Our results show that the differentiated phenotype in HCMV-speci�c CD8+ T cells was associated only with increased donor age whereas IFN-production in response to HCMV peptides was associated with HCMV replication, and also with recipient age and stem cell source.

Study Population.
Twenty-six HLA-A * 0201 patients who received allogeneic HSCT were recruited and peripheral blood samples were drawn at a median of 950 days aer HSCT (range 240-2436). Patients underwent HSCT at the Department of Haematology of the Reina So�a University Hospital (Cordoba, Spain).

HCMV Monitoring and Preemptive erapy.
Plasmatic HCMV viral loads were routinely screened using a Cobas Amplicor HCMV Monitor (Roche Diagnostics, Basel, Switzerland), a commercially available quantitative polymerase chain reaction (PCR) test with a detection limit of 600 copies of HCMVDNA/mL. e prospective monitorization protocol included two determinations per week during the �rst month or until discharge, and one determination per week until day +100 or +180 in patients with GVHD requiring high-dose steroids. HCMV replication was de�ned as the presence of any HCMV viral load in plasma over the limit of detection (>600 copies/mL).
Preemptive valganciclovir (Roche, Basel, Switzerland) was administered: (i) at the time of the �rst positive HCMV viral load in high-risk patients (unrelated donor transplant, steroid treatment) or in patients with a HCMV load ≥ 10.000 copies/mL in a single sample; (ii) at the time of a second positive sample obtained one week aer the �rst. Valganciclovir was administered orally in a dosage of 900 mg b.i.d. for 2 weeks (induction dose) followed by 900 mg qd until negativization of HCMV replication during 2 consecutive weeks (maintenance dose). e dosage was adjusted for creatinine clearance following standard recommendations. Valganciclovir was discontinued temporarily or substituted with foscarnet if necessary in patients with a neutrophil count < 0.5 × 109/L despite the administration of G-CSF.

Transplantation
Protocol. e conditioning regimen was myeloablative or reduced intensity conditioning protocol (RIC) in patients aged >50 years or with comorbidities. e myeloablative conditioning regimen consisted of hyperfractionated total body irradiation (13.2 Gy in 8 fractions) plus Cyclophosphamide (60 mg/kg/day for 2 consecutive days), Busulphan (0.8 mg/kg i.v. × 16 doses) plus Cyclophosphamide (60 mg/kg/day for 2 days) or ATG (rabbit, 2.5 mg/kg/day × 4 days) plus Cyclophosphamide (50 mg/ kg/day × 4 days). e reduced intensity protocols consisted of Fludarabine (30 mg/m 2 × 5 days) plus Busulphan (0.8 mg/kg i.v. × 10 doses) or plus Melphalan (70 mg/m 2 × 2 doses). Acute GVHD prophylaxis varied according to donor type and conditioning regimen intensity: recipients of a matched related transplant aer myeloablative conditioning received cyclosporin plus a short course of methotrexate while recipients of reduced intensity conditioning received cyclosporine plus mycophenolate mofetil. In addition, recipients of an unrelated donor gra in remission at the time of HSCT received rabbit ATG (ymoglobulin, Genzyme, Framingham, MA, USA) 6 mg/kg days −4 to −2.

INF-Production Using the QuantiFERON-HCMV
Assay. e QuantiFERON-HCMV test was performed according to the manufacturer's instructions (Cellestis Ltd., Melbourne, Australia). In brief, the assay consists of two steps: an overnight blood culture with HCMV CD8+ T-cell synthetic peptide epitopes and the subsequent quanti�cation of INF-production by an enzyme-linked immunosorbent assay (ELISA). Initially, 1 mL aliquots of heparinized whole blood were collected in three QuantiFERON-HCMV blood collection tubes that were shaken vigorously for 5 sec. e tubes contained either (i) a mix of 21 human cytomegalovirus (HCMV) peptide epitopes from a variety of HCMV proteins including pp65, IE-1, IE-2, pp50, and gB (1 g/mL of 21 different peptides), (ii) no antigens, only sterile phosphate buffered saline (negative control), or (iii) phytohemagglutinin (PHA; positive mitogen control). e tubes were incubated for 16-24 h at 37 ∘ C. Following incubation, supernatants were harvested and IFN-g levels measured (IU/mL) using a standard ELISA as per manufacturer's instructions. e results were calculated using Analysis Soware v 1.51 (Cellestis Ltd.). e cutoff value for HCMV reactivity was 0.2 IU/mL.

Statistical Analysis.
Statistical analysis was performed using SPSS soware for Windows v.18.0 and EPIDAT 3.1 soware. e population of HCMV-speci�c CD8+ T cells was expressed as a proportion of the total population of CD8+ T cells. e 2 test or Fisher exact test was used to assess the association between independent and dependent variables. A value < 0.05 was considered statistically signi�cant.

Patient Characteristics.
Twenty-six HLA-A * 0201 patients who received allogeneic HSCT for hematological malignancies at the Reina So�a University Hospital were studied. Samples were obtained aer lymphocyte recovery (1.000/10 9 /L), at least six months following HSCT (median 950 days; range 240-2436 days). e characteristics of the subjects in the study are summarized in Table 1. Median recipient age was 42 years old (range 16-68) and median donor age was 40 years (range 11-64). Fiy percent of the patients received a "nonmyeloablative" conditioning regimen. Fourteen patients received peripheral blood stem cells (53.8%) and 12 from bone marrow stem cells (46%). Fourteen HSCTs were donor (D) and recipient (R) HCMVseropositive (D+/R+), 10 were D−/R+ and 2 were D−/R−. Sixty-nine percent of patients received corticosteroids aer HSCT. Eleven patients (42.3%) experienced gra-versus-host disease (GVHD) and 15 (57.7%) had HCMV replications aer HSCT but none of them underwent HCMV disease.

Classi�cation o� Patients
According to the Phenotype o� HCMV-Speci�c CD�� T Cells. We used HLA-A * 0201(pp65) pentamers to study the frequency of the HCMV-speci�c subpopulation and the expression of CCR7, CD45RA, and CD28 on this subset in HSCT patients. Our results showed that the percentage of CD8+ T cells was 35.1% ± 14.8 and the percentage HCMV-speci�c over total CD8+ T cells was 1.36% ± 2.1. e phenotypic analysis showed that the mean percentage of HCMV-speci�c CD8+ T cells with naive phenotype (CD45RA+CCR7+) was 6.7% ± 11.3 and the mean percentage of CD28 expression on these cells was 30% ± 23.2.
Considering these values, patients were classi�ed into two groups: those having a "differentiated phenotype" if the HCMV-speci�c CD8+ T cells had values of CD45RA+CCR7+ naive cells ≤6.7% and CD28+ cells ≤30% and those having a "nondifferentiated phenotype" if they did not ful�ll these criteria. A representative example is shown in Figure 1.

�e Di�erentiated Phenotype o� HCMV-Speci�c CD�� T Cells in the Recipient Is Associated with Donor
Age. As shown in Table 2, donor age was the only factor signi�cantly associated with the presence of the "differentiated phenotype" in HCMV-speci�c CD8+ T cells in the recipient (OR = 12; ), whereas recipient's age, stem cell source or other HCMV-related factors were not statistically signi�cant. us, HSCT patients receiving stem cells from donors older than 40 years are 12 times more likely to have HCMV-speci�c CD8+ T cells with differentiated phenotype than patients graed from donors younger than 40 years old.

INF-Production in Response to HCMV Peptides Is Associated with Post-HSCT HCMV Replication, Recipients'
Age, and Stem Cell Source. e patients were divided into two groups according to INF-production in response to speci�c HCMV peptides using QuantiFERON-HCMV assay: "reactive" patients in whom the INF-production was higher than 0.2 IU/mL and "nonreactive" patients in whom the INF-production was lower. e results shown in Table 3 demonstrate a statistically signi�cant association with HCMV replication aer transplantation (OR = 11; 26), recipients' age (OR = 11; 26), and the stem cell source (OR = 17.5; 2 ). Hence, recipients who have experienced HCMV replication aer HSCT, those older than 40 and those graed with peripheral blood are more likely to harbour circulating effector HCMV-speci�c CD8+ T cells producing INF-. We did not �nd any other signi�cant difference although the association with donor HCMV serology was close to statistical signi�cance ( ). Although a multivariate analysis would be of help to better understand these associations, the relatively small sample size precludes doing it.

Discussion
In this work, we have studied clinical factors that have impact on T-cell phenotype and responses to HCMV in a crosssectional study of 26 HLA-A * 0201 HSCT recipients. Our results show that the "differentiated phenotype" (levels of HCMV-speci�c CD8+ T cells CCR7+CD45RA+ ≤6.7% and CD28+ ≤30%) is statistically associated only with donor age older than 40 years. A previous study [20] has been shown that HCMV-speci�c T cells aer HSCT mostly correspond to the expansion of HCMV-speci�c T cells from donors. e fate of these cells, which are adoptively transferred during HSCT, is determined by their characteristics in the donor rather than by other factors related to transplantation or virus reactivation. Furthermore, the authors showed that HCMV-speci�c T cells with a differentiated phenotype (CD27−CD57+) in the donor tended to increase in the recipient whereas less differentiated memory cells (CD27+CD45RO+CD57−) tended to decrease. In the study, the authors did not analyse factors associated with  10 0 F 1: �uantitative assessment of HCMV-speci�c T cell with "differentiated phenotype. " �uantitative assessment of HCMV-speci�c CD8� T cells using A2-pp�5 pentamer and monoclonal antibodies speci�c for CD8. e phenotype of HCMV-speci�c CD8� T cells was determined analyzing the expression of CCR7, CD45RA, and CD28. A representative example is shown. the percentage of naive or memory cells in either the donor or the recipient. However, we and others have shown that age is one of the major factors that determine the percentage of HCMV-speci�c CD8� T cells and their phenotype [21] in healthy individuals. us, the association between the "differentiated phenotype" and donors' age observed in our series li�ely re�ects the phenotype of the adoptively transferred donor cells. is observation is of interest since it has been shown that HCMV-speci�c T-cell immunity a�er HSCT is largely determined by the frequency and phenotype of HCMV-speci�c T cells from the donors [20,22], which are affected by ageing [17,21,[23][24][25].
In addition, we have studied IFN-production in response to HCMV peptides in these patients. Our results T 2: Univariate analysis of factors associated with "differentiated phenotype" in CD8+ HCMV-speci�c T cells.
HCMV reactivation aer HSCT is lower in patients with higher levels of HCMV-speci�c T cells with a terminally differentiated phenotype (CD45RO−CD62L−) in the donor gra [22]. Protection from HCMV reactivation has been associated to proliferation of HCMV-speci�c T cells [1] and especially to high levels of HCMV-speci�c CD8� T cells secreting INF- [26]. In kidney transplants receptors, Egli et al. [27] have shown differences in IFN-responses to HCMV-pp72 versus HCMV-pp65. e latter appeared to be better suited to interrogate the CD4 as well as the CD8 Tcell compartment in a ROC analysis, and associated a higher pp65-speci�c CD4 T-cell response with protection from CMV replication. Furthermore, protection against HCMV replication is associated to the presence of HCMV-speci�c T cells with that have the capacity to produce several cytokines in response to HCMV peptide (dual function IL2/IFN-or polyfunctional) whereas the presence of T cells that produced only IFN-was considered a sign of functionally exhausted T cells unable to efficiently control HCMV replication [28].
Our results showing the association of HCMV replication with IFN-production in response to HCMV peptides using the QuantiFERON-HCMV assay could suggest that IFNproduction is insufficient to control HCMV replication aer HSCT as suggested by Krol et al. [28], although it cannot be discarded that IFN-production is the consequence of the expansion and differentiation of IFN-producing HCMV-speci�c T cells in response to HCMV replication.
We also show that recipient's age is associated with IFNproduction in response to peptides. Patient's age has a profound signi�cance in HCMV replication as patients older than 40 have a higher probability of HCMV replication than younger patients, even if they had similar percentages of HCMV-speci�c T cells secreting IFN- [26].
Several studies have analysed differences in immune reconstitution by comparing different stem cell sources. Bone marrow and peripheral blood contain different ratios of naive and effector-memory T-cell subsets [29][30][31], which could explain the lower number of infections in recipients graed with peripheral blood stem cells [32]. HSCT using peripheral blood stem cells exhibited better recovery of HCMV-speci�c CD8� T-cell function 3 months aer transplantation compared to patients that received bone marrow stem cells [10], thus supporting that differences in INF-production by distinct T-cell subpopulations lead to different HCMV infection rates. In contrast, a recent study [33] reported a higher incidence of HCMV infection and disease one month aer peripheral blood stem cell transplantation that correlated with the lower functional capacity of HCMV-speci�c T cells, in particular with an increased proportion of HCMV-speci�c CD8 T cells that produced a single cytokine and a lower proportion of polyfunctional cells. Aer 3 months, however, HCMV-speci�c responses were similar between recipients of bone marrow or peripheral blood as the stem cell source. Our results showing that the use of peripheral blood associates with increased levels of IFN-production in response to HCMV peptides strongly suggest that peripheral blood contains higher proportions of HCMV-speci�c T cells with a capacity to release IFN-in response to HCMV than bone marrow. However, as indicated above, IFNproduction is insufficient to control HCMV replication. is incapacity to control HCMV could be due to the presence in these patients of HCMV-speci�c functionally exhausted T cells that produce only IFN-in response to HCMV as suggested by Krol et al. [28].

Conclusions
In summary, our results indicate that whereas the phenotype of HCMV-speci�c CD8� T cells aer HSCT is associated with donor age, IFN-production in response to HCMV peptides associates with HCMV replication episodes, recipient age, and the use of peripheral blood as the stem cell source. e association of IFN-production in response to HCMV peptides with HCMV replication episodes and peripheral blood as the source of stem cells supports that posttransplant HCMV replication induces the expansion of IFN-producing HCMV-speci�c T cells present in the donor's peripheral blood, contributing to the control of HCMV infection. However, it cannot be excluded that in vitro IFN-production is insufficient to control HCMV replication aer HSCT and that it might re�ect the presence of "monofunctionally" exhausted rather than "polyfunctional" effector-memory speci�c T cells [28]. It could be of interest to develop other tests easy to perform, economical and reliable to immunomonitor polyfunctional HCMV-speci�c T cells, which will likely correspond better to HCMV protection. In addition, since the observations reported here are derived from a cross-sectional study with a relatively small sample si�e, these �ndings cannot be generali�ed based on this study alone. us, it will be important to con�rm and extend these �ndings in a prospective study that include the analysis of HCMV-speci�c T cells before HSCT and other additional time points. Such a study would help clinicians to assess the risk of HCMV infection as well as immunotherapy strategies for the reconstitution of HCMV-speci�c T-cell immunity and the subsequent control of HCMV infection aer HSCT.