(2006) 52–59www.elsevier.com/locate/trim

Transplant Immunology 16

Clinical factors influencing T-cell receptor excision circle (TRECs) countsfollowing allogeneic stem cell transplantation in adults

Mónica Jiménez a, Carmen Martínez b,⁎, Guadalupe Ercilla b, Enric Carreras a,Álvaro Urbano-Ispízua a, Marta Aymerich a, Neus Villamor a, Nuria Amézaga b,

Montserrat Rovira a, Francesc Fernández-Avilés a, Emili Montserrat a

a Department of Hematology, Institute of Hematology and Oncology, Institut d'Investigacions Biomèdiques August Pi I Sunyer, IDIBAPS,Hospital Clínic, University of Barcelona, Villaroel 170, 08036 Barcelona, Spain

b Department of Immunology, Institut d'Investigacions Biomèdiques August Pi I Sunyer, IDIBAPS, Hospital Clínic, University of Barcelona, Spain

Received 17 January 2006; accepted 24 February 2006

Abstract

To ascertain the clinical factors involved in T-cell reconstitution after allogeneic stem cell transplantation (SCT), we evaluated serialassessments of lymphocyte subsets by flow cytometry and TRECs levels by quantitative PCR in 83 adult patients. Patient age >25 years,unrelated donor, CMV infection and acute graft-versus-host disease (GVHD) adversely affected CD3+ and CD8+ T-cell recovery after SCT(p<0.05). TRECs were low or undetectable during the first months after transplant and progressively increased thereafter. However, medianTRECs of patients did never achieve normal values compared to healthy donors (median follow-up 9months, range 2–42). Presence andseverity of chronic GVHD significantly affected TRECs counts: patients with chronic GVHD had lower TRECs than patients without GVHDat 9, 12 and 24months after SCT (p=0.002, p=0.022, p=0.015). Patients with limited chronic GVHD had higher TRECs compared to patientswith extensive GVHD (p=0.018). No relationship was observed between fungal or bacterial infections and TRECs. Nonetheless, CMVinfection was associated with lower TRECs (p=0.032). Our data support the concept that adult thymus contributes with a slow but continuousproduction of thymic T cells to immune reconstitution after SCT. Chronic GVHD is the main factor associated to a delay in TRECs countsrecovery.© 2006 Elsevier B.V. All rights reserved.

Keywords: TRECs; Immune reconstitution; Allogeneic stem cell transplantation; GVHD

1. Introduction

One of the more interesting debates in the field of immunereconstitution after allogeneic stem cell transplantation (SCT)concerns the contribution of the thymus to the T-lymphocyterepopulation of the patient. Complete reconstitution of T-cellimmunity after transplantation requires the generation of newnaïve T cells from the thymus [1]. Because the thymus isprogressively replaced by fat with age, thymus T-cell output hasbeen assumed to be severely limited in healthy adults. Thisphysiological involution of the thymus with age has beensuggested to be an important cause of delayed T-cell

⁎ Corresponding author. Tel.: +34 93 2275428; fax: +34 93 2275428.E-mail address: cmarti@clinic.ub.es (C. Martínez).

0966-3274/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.trim.2006.02.006

reconstitution after SCT in adults. Besides age, other factorssuch as chemoradiotherapy, graft-versus-host disease (GVHD)and immunosuppressive drugs may inhibit thymopoieticfunction after transplantation [2–4].

Until recently, thymopoiesis has been indirectly evaluated bymeasuring phenotypically naïve T cells in peripheral blood.Unfortunately, phenotypic markers are not reliable since naïve Tcells can expand extrathymically without stimulation [5] andmemory cells may spontaneously revert back to naïvephenotype [6]. Chest computed tomography measurement ofthymic volume has also been used to evaluate thymus statusbased on the assumption that its volume correlates with function[7,8]. Douek et al. first reported the use of T-cell receptorexcision circles (TRECs) as a method to measure thymicfunction [2]. TRECs are episomal excision products of the T-

Table 1Patient characteristics (n=83)

Age, median (range) (years) 35 (15–57)Sex, n (%)Male 40 (48)Female 43 (52)

Diagnosis, n (%)CML 29 (35)AML 22 (26.5)ALL 12 (14.5)AM 2 (2.4)MDS 9 (11)NHL 3 (3.6)MM 1 (1)CLL 4 (5)Others 1 (1)

Disease stage at allo-SCT, n (%)Good risk 52 (63)Poor risk 31 (37)

Donor, n (%)HLA matched sibling donor 57 (69)HLA matched unrelated donor 25 (30)HLA mismatched unrelated donor 1 (1)

Conditioning regimen, n (%)Cy+TBI 70 (84.4)BU+Cy 6 (7.2)Cy+TAI 2 (2.4)BEAM 2 (2.4)BU+Mel 2 (2.4)Cy+TBI+ATG 1 (1)

GVHD prophylaxis, n (%)CSP 17 (20.5)CSP/MTX 59 (71)CSP/MMF 3 (3.6)CSP/PD 4 (4.8)

Source of stem cells, n (%)Peripheral blood 55 (66)Bone marrow 28 (34)

T-cell depletion, n (%) 31 (37)Number of infused CD34+ cells×106/kg body receptorweight, median (range)

3.35 (1.1–14.7)

Number of infused CD3+ cells×106/kg body receptor weight,median (range)

16 (0.1–459)

CML, chronic myeloid leukemia; AML, acute myeloid leukemia; ALL, acutelymphoid leukemia; AA, aplastic anemia; MDS, myelodisplasic syndrome;NHL, non-Hodgkin's lymphoma; HD, Hodgkin's disease; MM, multiplemyeloma; CLL, chronic lymphocytic leukemia; SCT, stem cell transplantation;TBI, total body irradiation; Cy, cyclophosphamide; BU, busulphan; TAI,thoraco-abdominal irradiation; Mel, melphalan; ATG, anti-thymocyte globulin;BEAM, BCNU, etoposide, Ara-C, melphalan; GVHD, graft-versus-hostdisease; CSP, cyclosporine A; MTX, methrotrexate; PD, prednisolone.

53M. Jiménez et al. / Transplant Immunology 16 (2006) 52–59

cell receptor genes rearrangements that occur in maturingthymocytes. Intrathymic formation of a productive T-cellreceptor (TCR) α-gene requires deletion of the TCR δ-gene,which is positioned within the TCRα locus [9]. The TCRδ-geneis flanked by two TCRδ deleting elements, δRec-ψJα, whichpreferentially rearrange to each other, thereby deleting the TCRlocus [5]. The deleted TCR-gene remains present as anextrachromosomal circular excision product [10]. TheseTRECs do not replicate during mitosis and are thus dilutedduring cell division [11]. With real-time quantitative polymer-ase chain reaction (RT-PCR), TRECs can be detected andquantified, this providing a tool for identifying recent thymicemigrants and to estimate thymic output.

Several reports taking TRECs analysis as a quantitativemarker for thymus function have shown that T-cell neogenesiscontributes to immune reconstitution not only in children butalso in adult patients following SCT [3,4,12,13]. However,TRECs data from human clinical studies should be interpretedwith caution. TRECs counts can be affected by other biologicalparameters different to thymic output such as cell division,apoptosis of T cells, longevity of naïve T cells and intracellulardegradation [14]. Moreover, as mentioned above, the rate anddegree of immunologic reconstitution can be influenced by avariety of clinical factors, including patient age, conditioningregimen, stem cell source, T-cell depletion (TCD), type ofdonor, immunosuppressive therapy and graft-versus-host dis-ease (GVHD) [2–4].

In this study, we used a combination of T-cell phenotype andTRECs quantification to analyze T-cell reconstitution after SCTin a group of 83 adult patients from a single institution. Aunivariate and multivariate logistic regression model was usedto determine the impact on immune recovery of several clinicalvariables that potentially could affect thymic function.

2. Materials and methods

2.1. Patients

From January 1998 until November 2003, a total of 155 consecutive adultpatients received a myeloablative allogeneic SCT in our institution. Of these, 83patients were included in the present retrospective study. Patient exclusion wasdue to early death (less than 60days) after SCT (n=52), persistence of diseaseimmediately after SCT (n=4) and absence of optimal blood samples collectedfor analysis (n=16). Blood specimens were sequentially drawn from recipientsafter SCT for lymphocyte phenotypic analysis and for DNA isolation andstorage. Informed consent was obtained from patients using the protocols andforms approved by the institution's Ethical Committee.

2.2. Graft-versus-host disease (GVHD)

GVHD prophylaxis is summarized in Table 1. Acute and chronic GVHDwere diagnosed and graded according to the Seattle criteria [15]. Qualitativeinformation about GVHD target-organ involvement and the onset of symptomswere collected retrospectively.

2.3. Infections

Cytomegalovirus (CMV) antigenemia was determined periodically by theimmune alkaline phosphatase technique (monoclonal antibody pp65) in bloodsamples. Bacterial infection was defined as the microbiological identification of

a pathogen from culture from a sterile or non-sterile site. Viral infection wasdefined as the microbiological identification of a pathogen from culture or asviral detection by PCR. Fungal infection was defined according to the EORTC-MSG criteria [16].

2.4. Flow cytometry

Blood samples were obtained at 1 (n=50), 3 (n=45), 6 (n=38), 9 (n=32), 12(n=29), 24 (n=19), 36 (n=8) and 42 (n=2) months post-transplantation. Flowcytometry analysis of T- and B-lymphocyte subsets were performed as part ofthe standard follow-up of the patients after SCT in our institution. The analysisconsisted of quantification of total lymphocytes, CD3+, CD4+, CD8+, NK cells(CD16−CD56+) and CD19+ lymphocytes. Four-parameter flow cytometricanalysis was performed on a two-laser FACSCalibur flow cytometer (BectonDickinson Immunocytometry Systems [BIDIS], San Jose, CA). The samples

Table 2GVHD and infectious complications in recipients of allogeneic SCT

GVHD n

Acute 57 (69%)Chronic 31 (37%)

Infectious complicationsCMV 22CMV antigenemia 17CMV disease 5

Other viral infections 3Respiratory syncitial virus pneumonia 1Influenza virus pneumonia 1Viral hemorrhagic cystitis 1

Severe bacterial infections 27Gram-negative bacteriemia 14Gram-positive bacteriemia 9Mycobacterium tuberculosis 1Nocardia sepsis 1Pneumococcal pneumonia 2

Invasive fungal infection 11

GVHD, graft-versus-host disease; CMV, cytomegalovirus.

54 M. Jiménez et al. / Transplant Immunology 16 (2006) 52–59

were stained with the following monoclonal antibodies (MoAb): CD3 (Leu-3a)PerCP, CD8 (leu-2a) FICT, CD56 (leu-19) PE, CD45 (LCA) APC, CD19 (leu-12) FICT, CD4 PE. Normal values of each cell subset were determined from 12healthy donors.

2.5. Cell isolation and DNA extraction

Peripheral blood mononuclear cells (PBMNCs) were isolated by Ficoll-Hypaque density-gradient centrifugation from heparinized blood. CD3+ T cellswere isolated of PBMNCs by magnetic bead separation over columns using theMiniMACs multisort kit according to the manufacturer's instructions (MiltenyBiotec Inc., Sunnyvale, CA). For depletion of B cells, monocytes, NK cells,dendritic cells, early erythroid cells, platelets and basophils from PBMC, thesecells were indirectly magnetically labeled using a mixture of hapten-conjugatedCD19, CD11b, CD56, CD16 and CD36 antibodies and MACS MicroBeadscoupled to an anti-hapten monoclonal antibody isolation. With this technique, atleast 90% purity was achieved as determined by flow cytometry. DNA wasextracted from selected CD3+ cells using silica membranes (Quiamp Blood Kit,Qiagen, IZASA, Barcelona, Spain) according to the manufacturer's instructions.DNAwas quantified using standard UV-absorption at 260 nm twice at differentdilutions and stored.

2.6. TRECs analysis

Quantification of δRec-ψJα signal-joint T cell receptor excision circles(TRECs) was performed by RT-PCR using a LightCycler system (RocheMolecular Biochemicals, Mannheim, Germany) as previously described [17].In summary, PCR reactions were performed in a volume of 20μL with400 nmol/L of primers (5′-GTGACATGGAGGGCTGAAC, 3′-CTCTCCAAGCAAAATGGG), 200nmol/L (0,2μM) of detection probelabeled on its 5′ end with the acceptor fluorophore LightCycler RED640(5′LC Red640-AAAACCAGAGGTGTCACGATGGT), 100nmol/L of detec-tion probe labeled on its 3′ end with fluorescein (5′-CCACAGGAGTGGG-CACCTTTAC-FL), 100ng DNA and 4μL of LightCycler DNA Master PlusHybridization Probes (Roche Molecular Biochemicals). Conditions were95°C for 9min followed by 50 cycles of repeated denaturation (1s at 95°C),annealing (15s at 57°C) and enzymatic chain extension (25s at 72°C). Thehuman β-globin gene was used to normalize genomic DNA concentration.Samples were analyzed in duplicate or triplicate. TREC levels wereexpressed as absolute counts/μg DNA. The sensitivity of the assay was of2×10 copies.

2.7. Statistical analysis

Student's t-test with two-sided p-values, non-parametric Mann–Whitney U-test and chi-square test were used when indicated. Correlations were calculatedusing Spearman and Pearson correlation coefficients. Multiple linear regressionswere used to determine factors with possible influence on lymphoid and TRECsrecovery after SCT. Factors included in the analysis were: patient age, type ofdonor (sibling vs. unrelated), source of stem cells (peripheral blood vs. bonemarrow), TCD of the graft (yes vs. no), CD34+ cells dose infused with the graft(≤ vs. > median), acute and chronic GVHD (presence vs. absence), andinfectious complications. For the purpose of multivariate analysis, two periodsafter SCTwere considered: the early period post-SCT (1 to 3months, n=53) andthe late period post-SCT (6 to 12months, n=48). All statistical studies wereperformed using the SPSS 11.5 for Windows statistical software (StatisticalPackage for the Social Sciences 11.5, Chicago, IL).

3. Results

3.1. Patients

Demographic characteristics, diagnoses, conditioning regimensand parameters that may influence immune reconstitution after SCTare summarized in Table 1. All patients were conditioned withmyeloablative regimens. Thirty-one (37%) patients received TCDallografts from granulocyte colony-stimulating factor mobilized

peripheral blood stem cells (PBSC) and 52 (62%) receivedunmanipulated allografts (28 PBSC and 24 bone marrow stemcells). Median follow-up was 9months (range 2–42months). Twenty-nine patients presented treatment failure: 17 patients relapsed and 12patients died due to SCT complications. Infectious complications asdefined previously are summarized in Table 2. A total of 63 episodesof viral, bacterial or fungal infections were observed. CMV infectionoccurred in 17 patients and CMV disease in 5 patients after a medianof 63days (range 16–270) post-SCT. A total of 57 (69%) patientsdeveloped acute GVHD (grades II–IV in 28 patients) and 31 (37%)out of 72 patients at risk, chronic GVHD (15 limited and 16 extensivechronic GVHD).

3.2. Lymphocyte immunophenotypic analysis

3.2.1. Kinetics of recovery of lymphocytes subsets after SCTLymphocyte reconstitution after SCT was characterized by a rapid

recovery of NK cells and CD8+ T cells, and a slow recovery of CD4+ Tlymphocytes. Thus, levels of NK cells (CD16− CD56+) alwaysremained in the normal range after SCT, and normal values ofCD3+CD8+ T cells were achieved after a median of 3months. On thecontrary, CD3+CD4+ T-lymphocyte counts continued to be persistentlybelow healthy control levels until 18months post-SCT (Fig. 1). Incomparison to healthy donors, B lymphocytes (CD19+) achievednormal levels at 9months after SCT (Fig. 1).

3.2.2. Factors affecting lymphocyte reconstitutionNo differences were observed between patients according to age

except for a more rapid recovery of total CD3+ T cells and CD8+ T cellsin the late period of SCT in patients younger than 25years (p=0.059and p=0.03, respectively) (Table 4).

Type of donor significantly affected T- and B-cell reconstitutionafter transplantation. Thus, in univariate analysis, patients receivingunrelated SCT had lower total lymphocyte counts at 9 and 12months(p=0.016 and p=0.013, respectively), lower CD3+ counts at 12months(p=0.002), lower CD8+ counts at 6 and 12months (p=0.019 andp=0.006), and lower CD19+ counts at 3, 6 and 9months (p=0.023,p=0.024 and p=0.006) after SCT in comparison to recipients of siblingSCT (Table 3). Multivariate analysis confirmed these differences at the

Fig. 1. Median lymphocyte subset counts on 1 (n=41), 3 (n=41), 6 (n=40), 9 (n=30), 12 (n=21), 24 (n=21) and 36 (n=10) months post-SCT. Shaded area indicatesreference range, i.e., 10th to 90th percentiles of 12 healthy adult donors.

55M. Jiménez et al. / Transplant Immunology 16 (2006) 52–59

late period after transplantation for total, CD3+ and CD8+ lymphocytes(p=0.026, p=0.02 and p=0.004) (Table 4).

Source of stem cells (PBPC vs. bone marrow) and TCD of the grafthad little effect on lymphocyte reconstitution. Thus, except for a highernumber of total lymphocyte counts at 3months after transplantation inpatients receiving PBPC transplantation (p=0.009), no other differ-ences were observed. Recipients of unmanipulated grafts had higherlevels of CD19+ cells at 3 and 6months (p<0.001) and higher CD3+

and CD8+ T cells at 6months in comparison to TCD transplants inunivariate analysis (p=0.034 and p=0.04, respectively); however,multivariate analysis only showed a tendency for higher numbers ofCD8+ T cells in unmanipulated SCT recipients (p=0.08) (Table 4).

A dose of CD34+ cells greater than 3×106/kg body receptor weightwas associated with higher total lymphocyte and NK cells counts at1month (p=0.056 and p=0.012) and higher CD19+ cell counts at3months (p=0.046) in univariate analysis (Table 3), and with higherCD19+ cell counts in multivariate analyses at late period after SCT(p=0.051) (Table 4).

3.2.3. Effect of clinical events on lymphocyte reconstitutionIn multivariate analysis, acute GVHD was associated with lower

CD3+ and CD8+ cell counts (p=0.01 and p=0.03, respectively) andwith a trend to decreased CD19+ cell counts (p=0.087) (Table 4). Norelationship was observed between severe bacterial or fungal infectionsand lymphocyte reconstitution. However, the occurrence of CMVinfection was associated with decreased total lymphocyte, CD3+ andCD8+ T-cell counts during the first year post-SCT (p<0.05) (Table 3).Multivariate analysis confirmed this finding at early and late periodpost-SCT (p=0.09 and p=0.001 for total lymphocytes; p=0.073 andp=0.001 for CD3+ cells; and p=0.022 and p<0.001 for CD8+ cells,respectively) (Table 4).

3.3. TRECs analysis

3.3.1. TRECs levels in healthy individualsPrior to G-CSF administration, we examined TRECs levels in

PBMNCs and in sorted CD3+ cells from 12 adult healthy donors.

Median TRECs levels/μg DNA in CD3+ was 2.4×105 (range1.2×104–6.1×105).

3.3.2. TRECs analysis in CD3+ cells in SCT recipientsTRECs levels were low or undetectable in the first three months

post-SCT and progressively increased thereafter. Thus, mean count ofTRECs levels/μg DNA in CD3+ cells raised from 1.1×103 (range 0–3.5×104) on day 30 to 9×104/μg (range 0–6.1×105) at 12months aftertransplant (p=0.06). However, in comparison to normal donors,patients did not reach normal levels throughout the study (Fig. 2).Moreover, a considerable proportion of patients had undetectable levelsof TRECs at each time point after transplant and 28% of them still hadundetectable levels at 24months after SCT.

3.3.3. Factors affecting TREC levelsIt has been previously reported that increasing age negatively

correlates with numbers of phenotypically naïve T-cell and TRECscounts after transplantation. In our study, patients younger than25years significantly showed higher levels of TRECs in comparison toolder patients at early period after transplantation (p=0.026). However,multivariate analysis did not confirm this finding (Table 5).

No differences in TRECs levels were observed according to thetype of donor (sibling vs. unrelated), the source of stem cells (bonemarrow vs. peripheral blood) or TCD of the graft. In unmanipulatedrecipients, the dose of infused CD3+ cells did not correlate with TRECslevels at any time point post-SCT. Patients receiving a CD34+ cellsdose higher than 3×106/kg tended to have higher levels of TRECs atlate period post-SCT (p=0.068).

3.3.4. Effect of clinical events on TRECs levelsAcute GVHD did not affect TRECs levels in this study. In contrast,

the presence of chronic GVHD was associated with lower TRECslevels. In univariate analysis, patients with chronic GVHD had lowerTRECs levels than patients without chronic GVHD at 9, 12 and24months (p=0.002, p=0.022 and p=0.015, respectively). Multivar-iate analysis showed a trend to lower TRECs levels in patients withchronic GVHD (p=0.064) (Table 5). Moreover, the severity of chronic

Table 3Univariate analysis of lymphocyte subpopulations after allo-SCT

Age>25years

Unrelateddonor

TCD CD34+

>3×106/kg)GVHDa CMV

infection

Total lymphocyte1month ns ns ns 0.056 ns 0.0443months ns ns ns ns ns ns6months ns ns 0.033 ns ns <0.0019months ns 0.016 ns ns ns 0.00512months 0.049 0.013 ns ns ns 0.00624months ns ns ns ns ns ns

CD3+T cells1month ns ns ns ns ns ns3months ns ns ns ns 0.058 ns6months ns ns 0.034 ns ns 0.0039months ns ns ns ns ns 0.00212months 0.032 0.002 ns ns ns ns24months ns ns ns ns 0.036 ns

CD4+T cells1month ns ns ns ns ns ns3months ns ns ns ns ns ns6months ns ns ns ns ns ns9months ns ns ns ns ns ns12months 0.032 ns ns ns ns ns24months ns ns ns ns ns ns

CD8+ T cells1month ns ns ns ns ns 0.0263months ns ns ns ns ns 0.0176months ns 0.019 0.004 ns ns 0.0129months ns ns ns ns ns <0.00112months ns 0.006 ns ns ns 0.02824months ns ns ns ns ns ns

CD19+ T cells1month ns ns ns ns ns ns3months ns 0.023 <0.001 0.046 0.056 ns6months ns 0.024 <0.001 ns ns ns9months ns 0.006 ns ns 0.03 ns12months ns ns ns ns 0.026 ns24months ns ns ns ns ns ns

NK cells1month ns ns ns 0.012 ns ns3months ns ns ns ns ns 0.0576months ns ns ns ns ns ns9months ns ns ns ns ns ns12months ns ns ns 0.03 ns ns24months ns ns ns ns ns ns

TCD, T-cell depletion; GVHD, acute or chronic graft-versus-host disease; CMV,cytomegalovirus.a GVHD: acute GVHD (presence vs. absence) for 1 to 3months after

transplantation and chronic GVHD (presence vs. absence) for more than6months after transplantation.

Table 4Multivariate analyses of lymphocyte subpopulations at early (1–3months) andlate (6–12 months) periods post-SCT

Age>25years

Unrelateddonor

TCD CD34+

>3×106/kgGVHDa CMV

infection

Total lymphocytes1–3months ns ns ns ns ns 0.096–12months ns 0.026 ns ns ns 0.01

CD3+cells1–3months ns ns ns ns 0.01 0.0736–12months 0.059 0.02 ns ns ns 0.001

CD4+ cells1–3months ns ns ns ns ns ns6–12months ns ns ns ns ns ns

CD8+ cells1–3months ns ns ns ns 0.03 0.0226–12months 0.03 0.004 0.08 ns ns <0.001

CD19+ cells1–3months ns ns ns ns 0.08 ns6–12months ns ns ns 0.051 ns ns

NK cells1–3months ns ns ns ns ns ns6–12months ns ns ns ns ns ns

Regression analysis of ranks, adjusting the lymphocyte counts for patient age (≤vs. >25years); unrelated donor; T-cell depletion; history of CMV infection;GVHD, graft versus-host disease and dose of infused CD34+ cells (≤ vs.>3×106/kg body receptor weight).a GVHD: acute GVHD (presence vs. absence) for 1 to 3months after

transplantation and chronic GVHD (presence vs. absence) for 6 to 12monthsafter transplantation.

56 M. Jiménez et al. / Transplant Immunology 16 (2006) 52–59

GVHD appeared to have an effect over thymic output. Thus, patientswith limited chronic GVHD had similar TRECs levels than patientswithout chronic GVHD and higher TRECs levels compared to thosewith extensive chronic GVHD (p=0.018). This effect seemed to persisteven after the resolution of chronic GVHD and the suspension ofimmunosuppressive therapy. Thus, patients with history of extensivechronic GHVD tended to have lower TRECs levels than patients withhistory of limited chronic GVHD (p=0.068). Fig. 3 shows a composite

figure of the median and range levels of TRECs in CD3+ cells forpatients in relation to GVHD.

TRECs levels were also compared between patients who did or didnot develop severe infections. No relationship was observed betweeninvasive fungal infection or severe bacterial infections and TRECslevels. Nonetheless, the occurrence of CMV infection was associated inunivariate analyses with a tendency to decreased TRECs values(p=0.09 at 3months). Of note, the multivariate analyses showed lowerTREC levels at late period post-SCT in patients with history of CMVinfection (p=0.032) (Table 5).

4. Discussion

Delayed immune reconstitution following allogeneic SCTremains a major clinical problem, resulting in significanttransplant-related mortality from infectious complications.The recovery of immunity after SCT is a complex processthat depends on a large number of pre- and post-transplantfactors. In this study the main clinical factors associated withimpaired T-cell reconstitution were extensive chronic GVHDand CMV infection.

Several reports, using phenotypic lymphocyte analysis and/orTRECs quantification, have already shown the association betweenchronic GVHD and delayed T-cell reconstitution after SCT [4,17–23]. GVHD can affect T-cell recovery and TRECs levels in two

Fig. 2. TRECs levels/μg DNA CD3+ cells in healthy donors and patients afterSCT. The bottom represents the mean value. The top and the square though themiddle of box represent standard error. The whiskers on the bottom and toprepresent non-outliers range.

Fig. 3. TRECs levels/μg DNA CD3+ cells in patients with and without chronicGVHD at 9months after SCT. The bottom represents the mean value. The topand the square though the middle of box represent standard error. The whiskerson the bottom and top represent minim and maxim values.

57M. Jiménez et al. / Transplant Immunology 16 (2006) 52–59

different ways. First, GVHD could decrease thymopoietic outputthrough a direct attack on the thymic stroma by allogeneic effectorcells. Second, because GVHD is treated with immunosuppressivedrugs that may affect thymic function, the effect of GVHD on post-transplant T-cell recovery and TRECs levels could be due to suchdrugs. Our results show that not only activity but also the severity ofchronic GVHD has a deleterious effect on TRECs levels aftertransplantation. Thus, patients with limited chronic GVHD hadsimilar TRECs levels than patients without GVHD, but higherTRECs levels compared to those with extensive chronic GVHD.Since only patients with extensive chronic GVHD were treatedwith steroids and/or cyclosporine, it could be argue that theseimmunosuppressive agents are responsible of the delay of TRECsrecovery. Steroids and cyclosporine are known to cause thymocytesand lymphocytes apoptosis in vitro and in mice experimentalmodels; however, little is known of their effect on thymic output in

Table 5Univariate and multivariate analyses of factors affecting TRECs levels/μg DNAof CD3+ T cells at early (1–3months) and late (6–12months) periods post-SCT

TREC levels at earlyperiod post-SCT (n=53)

TREC levels at late periodpost-SCT (n=48)

Univariate ⁎ Multivariate ⁎⁎ Univariate ⁎ Multivariate ⁎⁎

Age>25 years 0.026 0.90 0.16 0.69Unrelateddonor

0.8 0.75 0.49 0.8

T-celldepletion

0.8 0.37 0.6 0.11

CD34+

>3×106/kg0.19 0.99 0.39 0.068

GVHDa 0.6 0.15 0.001 0.064CMVinfection

0.09 0.77 0.5 0.032

a GVHD: acute GVHD for the early period after SCT and chronic GVHD forthe late period after SCT.⁎ Mann–Whitney U-test.⁎⁎ Regression analysis of ranks, adjusting the lymphocyte counts for patientage (≤ vs. >25years); unrelated donor; T-cell depletion; dose of infused CD34+

cells (≤ vs. >3×106/kg body receptor weight); GVHD, graft-versus-hostdisease; CMV and history of cytomegalovirus infection.

humans [24–27]. Weinberg et al. recently reported no effect ofprophylactic immunosuppressive treatment on TRECs recovery inpatients without GVHD after cord blood cells transplantation.Moreover, we also observed that patients with extensive chronicGHVD had low TRECS levels even after the resolution of thiscomplication and the interruption of immunosuppressive drugs,suggesting the persistence of the thymic lesion induced by the allo-reaction. This is in agreement with a previous study in whichpatients with a history of prior acute or chronic GVHD hadpersistently low TRECs levels up to 10years after transplantation[4]. On the contrary, Poulin et al. observed that patients with pastepisodes of chronic GVHD had similar TRECs levels than thosewho never experienced this complication after SCT [23]. Authorsconclude that the impact of GVHD on thymopoiesis is reversible.However, no difference between limited and extensive chronicGVHDwas done in that study. The question of the relative roles ofGVHD and immunosuppressive drugs on immune reconstitutionremains open.

In agreement with a previous report, we observed asignificant association between CMV infection and decreasedtotal lymphocyte, CD3+, CD8+ Tcells and TRECs counts duringthe first year post-SCT [22]. However, the mechanisms involvedin this association are not well understood. On the basis ofavailable data, it is not possible to distinguish whether lowerTRECs counts result in higher risk of CMV infection oralternatively CMVinfection by itself or its treatment delay T-cellreconstitution after SCT. In our study, patients had CMVinfection after a median of 63days and only four presented CMVinfection after day 100. The temporal relationship between theearly period after SCT and the occurrence of CMV infection hasbeen well documented, and is thought to be related mainly to theCMV-specific CD8+ T cells deficiency [28–33]. However, animmunosuppressive effect of the CMV itself and/or and adverseeffect of antiviral therapy with ganciclovir cannot be rule out. Itis well known that CMV infection is one of the human viralinfections causing immunosuppression [34]. Two main mechan-isms are involved in this effect: first, CMV has a direct inhibitoryeffect on cytotoxic T lymphocytes and on monocyte and

58 M. Jiménez et al. / Transplant Immunology 16 (2006) 52–59

dendritic cells function; and, second, CMV is able to infect bonemarrow progenitor cells as well as stromal support cells causingmyelosuppression and immunosuppression [35–38]. Moreover,treatment of CMV with ganciclovir could also contribute todelay immune recovery. Ganciclovir may cause immunosup-pression due to its antiproliferative effect, either by reducingmonocytes and dendritic cells or by directly affecting lympho-cytes [39].

An accurate interpretation of these data has to take intoaccount that TRECs levels in peripheral blood are the result of adynamic process depending not only on thymic output ofTRECs bearing cells, but also on peripheral expansion anddeath of T cell, which decrease TRECs counts. Increasednumbers of cycling Ki67+ peripheral blood lymphocytes havebeen observed in patients with ongoing inflammatory processessuch as infections and acute GVHD. Combining TRECsquantification and Ki67 analysis, Hazenberg et al. reportedthat increased peripheral cell division had a negative effect onTRECs numbers [40]. Significant expansions of minorhistocompatibility antigen-specific cytotoxic T lymphocyteshave been demonstrated to occur during acute and chronicGVHD [41]. Apoptosis of lymphocytes is also known to play animportant role on lymphocyte homeostasis after SCT. Lin et al.recently reported that apoptosis was greater among CD4+ andCD8+ T cells taken from patients in the first three months afterSCT compared with healthy controls [42]. Together, theseobservations suggest that activation of T cells in vivo byinfections such as CMV or by alloantigens during chronicGVHD induces T-cell division or apoptosis that significantlycould contribute to decrease TRECs levels.

In conclusion, our data support the concept that adult thymuscontributes with a slow but continuous production of thymic Tcells to immune reconstitution after SCT. Extensive chronicGVHD is the main factor associated to a delay in TRECsrecovery and this effect persists even after the resolution ofGVHD. Further investigations are needed to clarify theobserved association between CMV infection with decreasedcounts TRECs and its clinical relevance.

Acknowledgments

This work was supported in part by grants from Institutd'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS),Red Temática del Cáncer Instituto de Salud Carlos III no.CO3/10, FIS PI020622 and PI02/0350 from the Fondo deInvestigaciones Sanitarias de la Seguridad Social, SpanishMinistry of Health, and SGR2001 00375 from Generalitat deCatalunya.

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