Journal of Medical Virology 70:399–403 (2003)

Reconstitution of Lymphocyte Populationsand Cytomegalovirus Viremia or DiseaseAfter Allogeneic Peripheral BloodStem Cell Transplantation

Antonio Gutierrez,1 Isabel Munoz,2 Carlos Solano,1 Isabel Benet,1 Concepcion Gimeno,2

Isabel Marugan,1 Marıa Dolores Gea,1 Javier Garcıa-Conde,1 and David Navarro2,3*1Department of Hematology and Medical Oncology, School of Medicine, University of Valencia, Spain2Department of Microbiology, School of Medicine, University of Valencia, Spain3Department of Microbiology Hospital Clınico Universitario, Valencia, Spain

Early reconstitution of lymphoid populationswas monitored by immunophenotyping in 57allogeneic peripheral blood stem cell (allo-PBSC)transplant patients either with or without cyto-megalovirus (CMV) viremia or disease. Cellcounts for total lymphocytes and CD4þ T cellsabove the percentile 60th at day 14 postransplantwere associated significantly with CMV viremia-free survival within 120 days after transplant.Recovery of total lymphocyte, CD3þ, and CD8þ

T-cell counts proceeded at a more rapid rate inCMV viremic patients than in nonviremic pa-tients, irrespective of whether preemptive treat-ment with ganciclovir had been prescribed.Significant expansion of CD8þ and CD8þ CD57þ

T-cell subsets was associated with recovery fromviremia and no progression to CMV disease.Immunophenotyping may provide useful infor-mation for the clinicalmanagementof CMV infec-tion in allo-PBSC transplant recipients. J. Med.Virol. 70:399–403, 2003. � 2003Wiley-Liss, Inc.

KEY WORDS: lymphocyte subpopulations;cytomegalovirus; allogeneicstem cell transplantation

INTRODUCTION

Cytomegalovirus (CMV) remains a significant causeofmorbidity andmortality inallogeneic peripheral bloodstem cell transplant (allo-PBSC) transplant recipients,despite the implementation of potent antiviral drugssuch as ganciclovir and the development of highly sensi-tive methods to detect CMV viremia [Broers et al.,2000]. The availability of novel laboratory markers thatwould allow prediction of development of CMV viremia,and progression to or recovery fromCMVdisease, wouldgreatly improve the clinical and therapeutic manage-ment of CMV infection in these patients. In this setting,

functional methods directed to assessing reconstitutionof specific CD8þ and CD4þ T-cell immunity againstCMV appear to be particularly suitable [Reusser et al.,1991;Li et al., 1994;Aubert et al., 2001;Cwynarski etal.,2001; Gratama et al., 2001]. Nevertheless, they arecostly or laborious, making their implementation inroutine laboratories unfeasible at the present time.

Immunophenotypic analysis of peripheral blood lym-phocytes by flow cytometry constitutes a simple andrelatively low-cost, although nonspecific, approach toassessing the overall degree of immune restoration aftertransplantation. Monitoring of certain lymphocyte sub-sets by immunophenotyping has been shown to provideclinically useful information during CMV infection inrenal allograft transplant recipients [Van den Berget al., 1992; Nordoy et al., 1999]. Little is known,however, about the potential value of this approach formanagement of allogeneic marrow or stem cell trans-plantation patients [Gratama et al., 1987; Einsele et al.,1993; Nishio et al., 2001; Storek et al., 2001]. To gainfurther insight into this issue, thekinetics of lymphocytepopulation reconstitution was investigated by immuno-phenotyping in allo-PBSC transplant recipients whodeveloped either HCMV viremia or disease and com-pared with that of patients who did not develop HCMVinfection.

MATERIALS AND METHODS

Patients and Clinical Management

Fifty-seven patients (40males and 17 females) under-going unmodified (n¼16), T-cell-depleted (n¼30), and

*Correspondence to: Dr. David Navarro, Department of Micro-biology, School of Medicine, University of Valencia, Blasco Ibanez17 Valencia, 46010, Spain. E-mail: david.navarro@uv.es

Accepted 10 February 2002

DOI 10.1002/jmv.10409

Published online in Wiley InterScience(www.interscience.wiley.com)

� 2003 WILEY-LISS, INC.

nonmyeloablative (n¼11) allo-PBSC transplantationbetween October 1994 and December 2000 at the BoneMarrow Transplantation Unit of the Hematology andMedical Oncology Department of the Hospital ClınicoUniversitario de Valencia (Spain) were included in thestudy. Informed consent for blood sampling wasobtained from all patients and donors. The researchprotocol was approved by the Committee on ClinicalInvestigation of the Hospital Clınico Universitario.Underlying diseases were acute lymphoid or myeloidleukemia (n¼23), non-Hodgkin’s lymphoma (n¼13),chronic myeloid leukemia (n¼7), multiple myeloma(n¼5), myelodysplastic syndrome (n¼3), solid tumor(n¼3), Hodgkin’s disease (n¼2), and chronic myelo-proliferativedisorder (n¼ 1).The conditioning regimenswere either BuCy (busulphan 16 mg/kg and cyclopho-sphamide 120 mg/kg) or total-body irradiation (12–13 Gy) plus cyclophosphamide (120 mg/kg). Patientsreceiving T-cell-depleted inoculum were given Cyclos-porin A (CsA) (1.5 mg/kg every 12 hr until day þ60) asgraft-versus-host disease (GvHD) prophylaxis, whilepatients receiving unmodified inoculum were givenCsA (1.5 mg/kg every 12 hr until day þ180) andmethotrexate (15 mg/m2 on day þ1 and 10 mg/m2 ondaysþ3andþ6). Patients received eitherHLA-identicalsibling transplants (n¼ 47) or a single-allele mismatch-related donor transplant (n¼ 9). Recipient/donor CMVserostatus, as determined by a commercial enzyme-linked immunosorbent assay (ELISA) (CMV IgG test,Biokit, Barcelona, Spain) was as follows: positive/posi-tive (n¼ 41), positive/negative (n¼8), negative/positive(n¼5), and negative/negative (n¼2). From hospitaladmission onward, patients were given standard bac-terial (ciprofloxacin 500 mg twice daily [bid], p.o.),fungal (fluconazol 200 mg bid, p.o.), and viral (acyclovir800 mg three times daily [tid], p.o.) prophylaxis. Inaddition, all patients received weekly intravenous (i.v.)immunoglobulins at a dose of 400mg/kg until dayþ100,and monthly thereafter until day þ360. Patients weredivided into three groups. Group I included 16 patients(median age 39.5 years; range, 21–63) with no virol-ogical evidence of CMV viremia, group II included 32CMVviremic patients (median age; 50 years; range, 18–63 years), and group III included 9patients (median age,53 years; range, 37–59) who developed CMV end-organdisease: pneumonitis (n¼ 6), colitis (n¼1) and pneumo-nitis plus colitis (n¼ 2). No statistically significantdifferences (P>0.05) between groups were found forthe age of patients, the nature of the underlying dis-ease, the type of transplant, the conditioning regimen,the GvHD prophylaxis, type of viral prophylaxis, andthe number of CD34þ cells or lymphocytes infused.Acute GvHD developed in 24 patients (grades I–II,n¼12; grades III–IV, n¼12). Eleven patients hadchronic GvHD. No significant differences betweengroups were found for acute or chronic GvHD incidencerate, grade, day of onset, and therapeutic management.Only HLA matching was found to be significantlyrelated with occurrence of CMV end-organ disease(P¼ 0.03).

Diagnosis and Management of CMVViremia and Disease

The patients were monitored weekly from day 0 toþ120by the following procedures: detection of infectiousvirus in blood (viremia) by culture of blood cells ontoMRC-5 or HFF cells (shell vial), detection of pp65 inblood leukocytes (antigenemia-AG), and detection ofCMVDNA in plasma (AMPLICOR, Roche Diagnostics),the latter since June 1997. These procedures werecarried out as described previously [Solano et al., 2001].A patient was diagnosed of having CMV viremia whenone ormore of the aforementioned tests proved positive.CMV pneumonitis was diagnosed on the basis of clini-cal condition, the presence of interstitial infiltrates onchest radiographs, and the histological demonstrationof CMV inclusions in tissue samples obtained at biopsyor necropsy. CMV colitis was diagnosed by demons-tration of CMV inclusions in intestinal biopsies. Pre-emptive treatment with ganciclovir (5 mg/kg/12 hr forthe first 15 days and 5 mg/kg/day thereafter) and in-travenous immunoglobulin (400 mg/kg/day, every 48 hrfor 15 days) was given after a single positive AG (anynumber of positive cells). Ganciclovir treatment wasadministered �15 days after the negative conversion ofthe AG. Patients with CMV disease were treated withi.v. ganciclovir (5 mg/kg, 2 times daily for 21 daysfollowed by 5 mg/kg/day, 5 days/week for 1 month) andi.v. immunoglobulin (400 mg/kg/day, every 48 hr for15 days and weekly thereafter for 1 month).

Lymphocyte Immunophenotyping

Blood specimens were drawn from patients andsubject to immunophenotyping at days 14, 30, 60, and90 posttransplantation. White blood cell (WBC) countsand WBC differential counts were determined on anautomated cell counter (Coulter). Cells were stainedwith fluorochrome-conjugated monoclonal antibodies(purchased from Becton Dickinson, CA) acquired on aFACSscalibur cytofluorometer (Becton Dickinson), andanalyzed by CellQuest. The following antibodies wereused: anti-CD45-fluorescein isothiocyanate (FITC),anti-CD3-FITC, anti-CD8-FITC, anti-CD14-phycoery-thrin (PE), anti-CD4-PE, anti-HLA-DR-PE, anti-CD16CD56-PE, anti-CD57-PE, anti-CD19-PE, and aPerCP-conjugated anti-CD8 antibody, which permit-ted enumeration of the following lymphoid populations:T cells (CD3þ), activated T cells (CD3þHLA-DRþ),helper/inducer T cells (CD3þCD4þ), suppressor/cyto-toxic T cells (CD3þCD8þ), natural killer (NK) cells(CD3�CD16þCD56þ), B cells (CD19þ) and CD8þCD57þ

T cells, whose functional phenotype remains to be firmlyestablished. Undiluted monoclonal antibodies (10 ml)were added to 100 ml of whole blood followed by anincubation of 15 min at room temperature. Red bloodcells were then lysed with Ortho lysis solution andwashed twice. Lymphoid populations subject to analysiswere gated on using log 90-degree and forward scattercharacteristics. The monoclonal antibodies anti-CD45and anti-CD14 served to gate out residual red cells and

400 Gutierrez et al.

monocytes respectively. The percentages obtained forthe different lymphocyte subpopulations were multi-plied by the number of absolute lymphocytes todetermine peripheral cell counts for each lymphocytesubset.

Statistical Analysis

Comparison between the three groupswas carried outby nonparametric tests (Mann-Whitney U-test andKruskal-WallisH-test) for continuous variables, as wellas by chi-square analysis and Fisher exact tests fornominal variables. Kaplan-Meier analysis was employ-ed to estimate viremia and disease-free survival withinthe first 120 days after transplantation. Differencesbetween event-free survival curves were tested forstatistical significance using the two-tailed log-ranktest. All tests were two-sided and P< 0.05 was consid-ered statistically significant.

RESULTS

Fifty-seven patients were followed up from day 0 today 120 after transplantation. Sixteen of these patientsremained free of CMV viremia during the study period.CMV viremia developed in 41 of the 57 patients at amedian of 30 days (range, 8–90 days) postransplant. Ofthese patients, 32 cleared it and 9 progressed to CMVdisease, diagnosed at amedian of 34 days (range, 16–82)after transplantation. Three patients recovered fromdisease and survived to the end of the follow-up period.

We first investigated whether lymphocyte immuno-phenotyping early after transplant couldhelp anticipatedevelopment of either CMV viremia or disease. Patientcell counts for lymphoid populations at day 14 post-

transplantation (time point at which most patients hadnot developed CMV viremia) were stratified (above orbelow the 60th percentile) and Kaplan-Meier estimatesof CMV viremia-free survival (VFS) and CMV disease-free survival (DFS) by day 120 after transplantationwere obtained. Cell counts above 200 total lymphocytes/ml (P¼ 0.01) and 79 CD4þ T cells/ml (P¼0.01) associatedsignificantly with viremia-free survival (Fig. 1). Neitherestimates for cell levels of activated (CD3þ HLA-DRþ)T cells (P¼ 0.80), CD8þ T cells (P¼0.34) nor estimatesfor B (P¼ 0.28) and NK cells (P¼0.86) showed statis-tical significance. Kaplan-Meier estimates of disease-free survival for any of the lymphoid populations subjectto analysis lacked statistical significance (not shown).

We next compared lymphocyte population cell countsat days 30, 60, and 90 after transplantation amongpatients with and without viremia. The former groupincluded both patients who received preemptive ganci-clovir and those who did not, but did not comprisepatients who went on to develop CMV disease. The dataare presented in Table I. Cell counts at days 30 and60 did not differ significantly among groups, althoughperipheral levels for CD8þ T cells, CD8þ CD57þ T cellsand activated T cells (CD3þ HLA-DRþ) tended to behigher in the former group at the latter time point. Cellcounts for total lymphocytes, CD3þ T cells (P¼ 0.05),CD8þ T cells (P¼ 0.01), and CD8þ CD57þ T cells (P¼0.03), but not for CD4þ T cells (P¼ 0.09), NK cells(P¼0.71), and B cells (P¼ 0.15), were found to be signi-ficantly higher at day 90 in patientswho developedCMVviremia than in patients who did not.

Since ganciclovir has been shown to blunt recoveryof specific immunity against CMV [Li et al., 1994] wewondered whether repopulation of different T-cell

Fig. 1. Kaplan-Meier estimates of cytomegalovirus (CMV) viremia-free survivalwithin the first 120 daysafter allogeneic stem cell transplantation by CD4þ T lymphocytes cell counts at day 14 posttransplant.

CMV in Allogeneic Stem Cell Transplantation 401

subsets would be affected by preemptive treatment withganciclovir. At day 60 after transplantation, peripherallevels of CD8þ T cells were significantly lower (P¼0.04)in treated than in untreated patients. No statisticallysignificant differenceswere observed at day 90 for any ofthe T-cell subpopulations subject to analysis (notshown).

We then compared cell counts among viremic pa-tients with and without CMV disease. Comparisonswere carried out at days 60 and90after transplantation,time points at which 5 and 4 patients, respectively, whohad developed CMV disease remained alive. The dataare shown in Table I. Significantly lower peripheralcell counts for CD3þ T cells (P¼0.04), CD3þHLA-DRþ

T cells (P¼0.04), CD8þ T cells (P¼ 0.04) and CD8þ

CD57þ T cells (P¼0.02), were found on day 60 in pa-tients with CMV disease than in viremic patients whohad not progressed to disease. No significant differenceswere observed, for total lymphocytes (P¼0.06), CD4þ

T cells (P¼ 0.21), NK cells (P¼ 0.07), and B cells(P¼ 0.44). At day 90, cell counts for all populationsexamined tended to be higher in the latter patients,although statistical significance was not reached.

DISCUSSION

Several findings with potential clinical interest arisefrom this study. First, CMV viremia-free survival up today 120 after transplantation was associated signifi-cantly with relatively high peripheral blood levels fortotal lymphocytes andCD4þT cells at day 14, indicating

that immunophenotyping early after transplant mayhelp identify patients at highest risk of developing CMVviremia. Diminished CD3þ T and CD4þ T-cell recoverywithin the first 3weeks after CD34þ selected autologousPBST has also been linked with an increased risk ofCMV viremia [Nishio et al., 2001]. It was reportedrecently that episodes of abortive subclinical CMV in-fections, even in the absence of detectable viremia,might be associatedwith an increased predisposition forinvasive and fungal infections due to the immunosup-pressive potential of CMV [Garrett Nichols et al., 2002].In this setting, patients might benefit from early anti-CMV treatment guided by immunophenotyping. Even-tual progression to CMV disease could not be predictedby immunophenotyping early after transplantation;Nevertheless, the small number of patients with CMVdisease included this study does not allow to dismiss thevalue of immunophenotyping for that purpose; in fact,other studies [Einsele et al., 1993; Storek et al., 2001]conducted in allogeneic bone marrow transplant re-cipients have demonstrated a significant correlationbetween poor recovery of CD4þ T cells early after trans-plantation and high risk of developing CMV disease.

Second, repopulation of T cells, particularly CD8þ Tcells, proceeded at a faster rate in patients whodeveloped viremia, and cleared it thereafter, than inpatients in whom viremia was not detected; this effectwas noticeable even in patients who had received pre-emptive ganciclovir-which was shown to slow downrecovery of CD8þ T cells. A similar phenomenon hasbeen reported to occur in allogeneic bone marrow

TABLE I. Peripheral Cell Counts for Different Lymphocyte Populations in Allogeneic StemCell Transplant Recipients Who Developed Either Viremia or Disease

Cell population/dayafter transplant

Study groups/median cells/ml (range)

No CMV viremia(n¼ 16)

CMV viremia(n¼ 32)

CMV disease(n¼ 9)

Total lymphocytesþ60 730 (90–2190) 600 (195–3500) 300 (70–400)þ90 520 (160–950) 900 (310–4700) 680 (30–2750)

CD3þ

þ60 290 (37–1839) 379 (64–1678) 76 (9–334)þ90 359 (30–829) 677 (149–3821) 218 (11–2321)

CD3þHLA-DRþ

þ60 130 (8–1021) 258 (14–1186) 9 (0–179)þ90 152 (21–441) 374 (8–2514) 85 (2–1318)

CD3þCD4þ

þ60 145 (10–479) 112 (26–1376) 65 (3–128)þ90 123 (18–340) 221 (59–1773) 49 (4–394)

CD3þCD8þ

þ60 242 (27–1376) 267 (79–1334) 125 (20–226)þ90 194 (58–435) 486 (156–1313) 335 (8–1018)

CD8þCD57þ

þ60 77 (5–868) 117 (5–688) 23 (4–33)þ90 71 (6–256) 238 (44–885) 77 (7–667)

CD�CD16þCD56þ

þ60 227 (47–607) 126 (4–2763) 134 (52–215)þ90 125 (20–359) 150 (24–847) 283 (9–390)

CD19þ

þ60 5 (0–46) 6 (0–97) 1 (1–4)þ90 6 (0–33) 10 (0–201) 8 (0–70)

CMV, cytomegalovirus.

402 Gutierrez et al.

transplantation aswell as in renal transplant recipients[Gratama et al., 1987; Van den Berg et al., 1992; Nordoyet al., 1999]. Activation and expansion of CMV-specificCD8þ T-cell clones driven by CMV replication [Reusseretal., 1991;Li et al., 1994;Aubert et al., 2001;Cwynarskiet al., 2001; Gratama et al., 2001] may account for thesedata. In agreement with a previous study [Dolstra et al.,1995], amajor fraction of CD8þT cells consisted of CD8þ

CD57þ cells, which appear to represent an in vivoactivated subpopulation with cytotoxic activity [Dolstraet al., 1995]. On the basis of our data, eventual recoveryfrom a CMV viremia episode might be inferred bymonitoring peripheral levels of CD8þ CD57þ T cells,which in addition, may prove useful for determining theduration of preemptive treatments with ganciclovir.

Third, viremic patients who had progressed to CMVdisease displayed significantly lower peripheral countsfor CD3þ T cells, CD3 HLA-DRþ T cells, CD8þ T cells,andCD8þCD57þT cells, but not for CD4þT cells, at day60 after transplantation than those who had not, whichprobably reflects the fact that viremic patients unable tomount an appropriate effector T-cell response againstCMVare likely to go on to developCMVdisease [Reusseretal., 1991;Li et al., 1994;Aubert et al., 2001;Cwynarskiet al., 2001; Gratama et al., 2001]. Thus, frequentmonitoring of the above-mentioned lymphocyte sub-populations by immunophenotyping in CMV viremicpatients might prove useful for management of allo-PBSC transplant recipients as it may help anticipateprogression to CMV disease. In this sense, it could be avaluable parameter, complementary to virological mar-kers, for guiding initiation of preemptive therapy. Itwould also be of interest to determine whether recoveryfrom CMV disease could be predicted by immunophe-notyping; this issue could not be addressed given thescarce number of patients with CMV disease includedin the study.

In summary, the data suggest that serial analysis oflymphoid populations by immunophenotyping mayprovide useful information for management of CMVinfection in allo-PBSC transplant recipients; largerstudies are nevertheless needed to define the clinicalutility of this approach. A prospective study addressingthis issue is under way.

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