Delayed and/or insufficient T cell recovery post hematopoietic stem cell transplantation (HSCT) leads to an increased risk of morbidity and mortality. We evaluated thymic function and its association with T cell regeneration post HSCT and identified factors involved in the process among pediatric stem cell transplant recipients. T cell regeneration in 66 pediatric patients was prospectively followed by naive T cell phenotyping, measuring of T cell receptor excision circles (TRECs) and expression of Foxp3 by regulatory T cells for the first 18 months post HSCT. TRECs were lower pre-HSCT in children with a malignant than non-malignant primary disease or immunosuppressed controls (P=0.001). Naive T lymphocyte reconstitution and thymic recovery were slow in the recipients of allogeneic stem cell grafts post HSCT. Infections caused by herpesviruses had a prognostic impact on mortality. Children with low TRECs had a high mortality (P=0.05) and low TRECs were also associated with extensive chronic graft-versus-host disease from 6 months onwards. Low amount of Foxp3 pre-HSCT was associated with an increased mortality post HSCT (P=0.03). Our study indicates an association between impaired T cell regeneration and thymic dysfunction and the clinical post transplant complications in pediatric allogeneic stem cell transplantation.
T cell recovery, as a multifaceted process including recovery of the thymic function and that of the regulatory T cell compartment, plays a key role in the clinical recuperation of patients post hematopoietic stem cell transplantation (HSCT).1, 2, 3 Immunodeficiency post allogeneic stem cell transplantation is, on its part, associated with significant morbidity and mortality.4, 5
T lymphocytes are generated through two different pathways: thymus dependent and independent.6, 7, 8, 9, 10 Particularly in the hematopoietic stem cell transplantation setting, peripheral expansion of T cells can contribute significantly to the composition of the T cell compartment post HSCT.11 As additional factors, radiotherapy12 and graft-versus-host disease (GVHD)13, 14, 15, 16 post HSCT and thymic involution as a part of ageing have a negative impact on thymic function.15, 17, 18
Thymic function has been assessed by imaging and analysis of T cell subtypes in blood but more recently also by measuring TRECs (T cell receptor excision circles). Quantitative measurement of TRECs using PCR is assumed to reflect thymic output. However, persistence of naive T cells and TREC dilution in peripheral blood by cell division complicate the interpretation of TREC data as a measure of recent thymic output.14, 17
Previously published studies report on a correlation between TRECs and the frequency of naive CD4+ T cells in blood among pediatric and adult recipients of HSCT suggesting that most naive T cells are processed in thymus.16 Lewin15 reports on a faster thymic recovery post HSCT among children indicating that the high residual thymic activity of early childhood might allow for a rapid regeneration of T cells. The level of TRECs correlates negatively with chronic GVHD (cGVHD) in most studies,13, 14, 15, 16, 19 but conflicting results have also been reported.18, 20 Data is also indicative of an association between low TREC levels and post transplant infections.14, 15, 19
Regulatory CD4+CD25+ T cells play a key role in maintaining tolerance to self antigens and inducing non-responsiveness to alloantigens.21 Foxp3 (forkhead boxp3) gene encodes a transcription factor and is identified as a regulatory gene required for the development and functional activity of CD4+CD25+ regulatory T cells.22 Foxp3-transduced T cells have also been observed to control rejection of an allogeneic graft.23 Decreased expression of Foxp3 is associated with ongoing GVHD and increased expression with resolution of cGVHD in adults.20, 24 Also Foxp3 expression is reported to correlate with TREC levels in adults following HSCT,24 but no data on pediatric patients have been published.
The aim of this prospective study was to evaluate thymic function and T cell regeneration post HSCT and identify factors involved in the process among pediatric stem cell transplant recipients using analyses of T cell subtypes and TREC levels in peripheral blood as indicators of thymic function and Foxp3 expression as a marker of regulatory T cells.
Patients and methods
A total of 71 patients underwent HSCT between 06/2001 and 01/2004 at the Hospital for Children and Adolescents, University of Helsinki, Finland. Five patients were excluded from the study due to low weight (weight<10 kg) and the rest (66) (93% of all transplanted; 44 boys and 22 girls) enrolled following parental informed consent.
Thirty-one received their grafts from matched unrelated donors (URD) and 20 from HLA-identical siblings. The key characteristics of the allogeneic patients are given in Table 1.
Fifteen children given autologous stem cell rescue (median age 5 years, range 1–17 years) following high-dose chemotherapy were used as controls. Six of the 15 were administered multiple (2–3) autologous grafts. One-third of the autologous recipients had neuroblastoma and another third rhabdomyosarcoma. One-third of the recipients of autologous stem cell rescue had TBI in their conditioning. As an additional set of controls, children (a total of eight, median age 8, range 6–13 years) with either minimal change glomerulopathy or focal segmental glomerulosclerosis were enrolled into the study. One half of these non-transplanted, immunosuppressed controls had cyclosporin monotherapy and the rest cyclosporin with steroids at the time of analysis (median duration of cyclosporin 1.5 years, range 0.6–6.0 years and median duration of steroids 2.2 months, range 1–4 months).
T cell depletion was not performed on any of the allogeneic grafts. Only leukocyte-depleted and irradiated blood products were used. Fifty-seven of the 66 transplanted children received subcutaneous G-CSF at a dose of 5 μg/kg daily from day +1 until an ANC of 1000 × 106/l. No routine antiviral prophylaxis was employed. Eight allogeneic recipients received antilymphocyte globulin (ALG) as a part of their conditioning.
Neutrophil engraftment was defined as an ANC of >500 × 106/l. Thrombocyte engraftment was defined as a platelet count of above >30 × 109/l for three consecutive days without transfusion.
Routine GVHD prophylaxis consisted of daily cyclosporin (for 6–18 months) and three or four doses of methotrexate (intravenous). Acute GVHD (aGVHD) was graded according to the Glucksberg-Seattle criteria and clinically significant aGVHD defined as a grade II–IV disease. Initial therapy with prednisolone (2 mg/kg/day) was started for grade II (gut or liver) or grade III (skin) aGVHD and high-dose 5-day pulses of methylprednisolone (0.25–1 g/day in divided doses) employed for grades III–IV aGVHD and in some cases for extensive cGVHD. In addition to cyclosporin and prednisolone, azathioprine (n=16), mycophenolate mofetil (n=13), oxyclorin (n=4) and thalidomide (n=6) were used for extensive cGVHD.
Serology for CMV was tested before transplant. At weekly intervals after HSCT, throat swabs were analyzed for herpes simplex virus (HSV) and blood samples taken for CMV. Viral reactivation was defined (and pre-emptive therapy for CMV initiated) when reactivation was verified as the isolation of a virus antigen or virus itself from throat (HSV) or skin lesions (VZV) or nucleic acid (PCR, quantitative) detection in the blood (CMV). Disease was defined as reactivation combined with signs of organ involvement. Sixteen patients were considered at risk for CMV-related problems (donor negative and recipient positive serology) and 12 of them did receive viral prophylaxis.
The study was approved by the Institutional Review Board of the Helsinki University Hospital.
Blood samples were drawn once pretransplant and once every 3 months during the first year and at 18 months post transplant. The immunosuppressed, non-transplanted control children were tested only once. PBMC were separated by gradient centrifugation and TriReagent (Molecular Research Center, Cincinnati, OH, USA) used to extract both DNA and RNA.
Before reverse transcription (RT) RNA was treated with DNase in order to remove possible remnants of contaminating DNA. This treatment was performed with DNA-free (Ambion, Austin, TX, USA) following manufacturer's instructions. RT-reaction was performed with High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocol. RNA concentration in each reaction was 40 ng/μl and an RNase inhibitor (Applied Biosystems) added at 1 U/μl. Reactions were primed by random hexamers and Multiscribe RT enzyme employed with both being provided with the kit.
Quantification of TRECs
Quantification of thymic signal-joint TCR delta excision circles was performed by real-time quantitative PCR (ABI PRISM 7000; Applied Biosystems) according to Douek et al.17 and Talvensaari et al.25 A standard was created by cloning the signal-joint fragment in pCR2.1A using the TA cloning kit (Invitrogen, Groningen, The Netherlands). TRECs were determined by quantitative PCR on genomic DNA from PBMCs using an additional set of primers and a probe to albumin to normalize for the genomic copy number. The amount of TREC and albumin copies was calculated by including a dilution series of B-LCL cell line (Cox) genomic DNA in each PCR experiment (102–107 copies/well for TREC and 40–5000 ng/well for albumin). The TREC values were corrected for the percentage of CD3+ cells in the sample and expressed as the numbers of TREC/μg CD3+ cell DNA.
Quantification of Foxp3
To quantitate the amount of Foxp3 mRNA in samples TaqMan Gene Expression Assays (Hs00203958 m1 for Foxp3, Hs99999905 m1 for GAPDH) and ABI PRISM 7000 Sequence Detection System were employed as recommended by the manufacturer (Applied Biosystems). Real-time PCR reaction components and PCR conditions were as recommended by Applied Biosystems.
All real-time PCR amplifications were done in triplicate and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as an endogenous control gene for normalization. The same standard curve, diluted from total RNA from peripheral blood mononuclear cells of healthy blood donors, and non-template controls were included in every run.
To calculate the amount of Foxp3 mRNA in a sample the relative standard curve method was used (Applied Biosystems: User Bulletin #2). Briefly, input amounts were calculated using standard curves and relative amounts obtained by normalizing for the endogenous control. These results were proportioned to standards and then depicted as manifolds of them (cf. user bulletin #2 at ABIs website).
For flow cytometry cells in EDTA samples were stained using the whole blood lysis technique, and analyzed on FACSCalibur using the CellQuest software (Becton Dickinson, San Jose, CA, USA). The following monoclonal antibodies were used: anti-CD3 (clone SK7), -CD4 (SK3), -CD8 (SK1), -CD16 (B73.1), -CD45 (Hle-1), -CD45RA (L48), -CD45RO (UCHL-1), -CD56 (NCAM 16.2) and -CD27 (LI28), all from Becton Dickinson Inc. (Becton Dickinson). To establish the relative and absolute numbers in each lymphocyte subset MultiTEST CD3 FITC/CD8 PE/CD45 PerCP/CD4 APC and CD3 FITC/CD16CD56 PE/CD45 PerCP/CD19 APC reagents and TruCOUNT Tubes (Becton Dickinson) were used according to manufacturer's instructions.
Naive T lymphocytes were measured and expressed as the absolute number of CD3+CD45RA+CD27+ T cells in peripheral blood and memory/active T lymphocytes given as the absolute number of CD3+ T cells following subtraction of the number of the naive T cells.
The data was analyzed using the Statistical Package for Social Scientists (SPSS), version 12.0 for Windows.
Normal distribution was verified using the Kornikow–Smirnow test. Correlations were calculated using the Spearman's rank correlation coefficient (r-values). The Mann–Whitney–Wilcoxon rank-sum test (P-values) and multivariate logistic regression analysis were used in statistical analyses. Also the Cox survival model was used. Ninety-five percent confidence intervals (95% CI) were calculated when appropriate. Values of P<0.05 and r greater than 0.3 or less than –0.3 were considered significant.
Naive T lymphocytes and TRECs before transplant
The absolute count of naive T lymphocytes (CD3+CD45RA+CD27+) in the allogeneic recipients before transplant (a median of 0.20 × 109/l, CI 0.12–0.29) was low. In only 9/47 cases (19%, five sibling and four URD) were the counts at the same level as that of the immunosuppressed, non-transplanted controls (a median of 0.76 × 109/l, CI 0.47–0.86). However, the recipients of autologous stem cell rescue (a median of 0.05 × 109/l, CI 0.01–0.18) had even lower counts of naive T cells before transplant (P=0.02) (Figure 1).
The TRECs before transplant in the allogeneic group (a median of 459 TREC/μg CD3+ cell DNA, CI 58–873) were also low. In only 8/45 cases (18%, four sibling and four URD) were TRECs at the same level as among the immunosuppressed, non-transplanted controls (a median of 2849 TREC/μg CD3+ cell DNA, CI 780–7753). The recipients of allogeneic stem cell grafts (a median of 459 TREC/μg CD3+ cell DNA, CI 58–873) tended to have higher TRECs before transplant than those of autologous stem cell rescue (a median of 132 TREC/μg CD3+ cell DNA, CI 1–3939) (Figure 1).
Boys had lower TRECs before transplant than girls both in the whole group (P<0.01) as well as among the children with leukemia as a primary disease (P=0.004).
Naive T lymphocytes and TRECs post HSCT
The regeneration of naive T lymphocytes in the recipients of allogeneic stem cell grafts was slow. The absolute number of naive T cells in the allotransplanted children reached that of the immunosuppressed, non-transplanted control patients at 6 months in 1/51 cases (2%, sibling), at 9 months in 6/38 cases (16%, three sibling and three URD) and at 18 months in 16/32 cases (50%, 10 sibling and six URD). The recovery process in the recipients of autologous stem cell rescue was similar during the first 6 months post HSCT, but faster thereafter (Table 2).
Thymic recovery (TREC values) was gradual and bore resemblance to the naive T lymphocyte recovery. At 6 months 1/51 case (2%, sibling) and at 18 months 11/31 cases (35%, six sibling and five URD) had reached the level of the immunosuppressed, non-transplanted controls. However, within the allogeneic group the recipients of sibling stem cell grafts had higher naive T cells (P=0.02) and TRECs (P=0.03) at 6 months post HSCT than those of URD grafts (Figure 2).
Naive T cells (CD3+CD45RA+CD27+) correlated positively with TRECs (0.37<r<0.59) from 6 months onwards post HSCT. Also there were significant negative correlations post HSCT between patient age and both naive T cells (−0.60<r<−0.35) and TRECs (−0.44<r<−0.32) from 6 months onwards.
Factors associated with TRECs
Thirteen children developed CMV-reactivation (10/13 within 2 months post HSCT) and three of them had a fatal outcome (2/3 received antiviral prophylaxis). Seven children suffered from HSV stomatitis, mostly (six children) within 3 months post HSCT. Fourteen had VZV of the skin and again mostly within 3 months post HSCT. Herpesvirus (CMV, HSV and VZV) infections were not reflected in the levels of TRECs, Foxp3 or naive T cells. Memory/active T cells increased faster in the recipients with the herpesviral infections than among those without (Table 3). The recipients with an early herpesvirus infection(s) had more events later on than those without: 9/25 had a relapse (P=0.02, R2=6.94, CI (1.30–37.01), 6/25 treatment-related mortality (TRM) and 8/25 had non-lethal, but severe GVHD (Gr II–IV).
aGVHD grade II–IV was observed more often in the URD than sibling group (74 vs 30%, P=0.05) and 66% of those with aGVHD later developed cGVHD (Table 1). aGVHD did not associate with the level of TRECs or naive T cells. However, extensive cGVHD associated with low TREC levels (P<0.001) and low naive T cells (P=0.002) from 6 months onwards (Figure 3).
The regeneration of naive T lymphocytes in the recipients of allogeneic stem cell grafts with cGVHD was slower than in those without. Thymic recovery post HSCT parallelled the speed of recovery of the naive T cells: at 18 months 63% (10 children) of recipients without cGVHD reached the level of immunosuppressed, non-transplanted controls, while this was the case in only one child with cGVHD.
The overall mortality was 36% (24 cases): one-fourth autotransplanted and the rest allotransplanted (19 cases). The cause of death was relapse in 50% of the cases. Half of the patients with TRM had an identified herpesvirus infection(s) in their final stage. TRECs and the naive and memory/active T cells were lower among those with TRM during the first 6 months post HSCT (Table 3).
Foxp3 before transplant
A low amount of Foxp3 transcripts pretransplant associated with an increased mortality post transplant and appeared to be an independent factor (univariate P=0.03 and multivariate P=0.03; R2=0.25, CI 0.07–0.89), when the age of the recipient, the use of TBI, primary disease, aGVHD and viral infections were considered.
Foxp3 post HSCT
Clinically significant aGVHD (maximum severity) was associated with a low Foxp3 expression at 3 months (P=0.01, without aGVHD a median of 0.89 Foxp3/GAPHD transcripts, CI 0.36–1.29 and with aGVHD a median of 0.43 Foxp3/GAPHD transcripts, CI 0.16–0.64). Extensive cGVHD was associated with low Foxp3 post HSCT at 9 months (Figure 4). There was no significant difference between the recipients of sibling and unrelated grafts in Foxp3 expression post transplant (Figure 5).
In this prospective study focusing on pediatric recipients of allogeneic stem cell grafts we show that the regeneration of naive T lymphocytes and thymic recovery are slow. The delayed immune reconstitution is associated with an increased risk of clinical complications such as extensive cGVHD and a higher mortality.
Our study and those previously published indicate that thymic dysfunction exists even before transplant among stem cell graft recipients with a malignant disease and following a conventional chemotherapy.19, 26, 27 In our material the thymic output of naive T cells as well as TREC levels were lower just before transplant among children with a malignant disease. This may be a result of the underlying, and in many cases malignant lymphoid, disease and its therapy.28
We emphasize, that in the interpretation of the TREC results an important bias has to be recognized. Peripheral expansion of the T cell pool in the recipient during for example infection(s) may have diluting effect on the TREC levels. However, in our material the recovery of the TRECs paralleled that of naive T cells post HSCT.
The kinetics of thymic recovery (naive T lymphocytes and TRECs) post HSCT demonstrated two different phases, that is a shutdown followed by a rebound.26, 29 In our data, the lowest values appeared to coincide in the sibling and unrelated groups during the first 3 months post HSCT. As previously reported,29 the initiation of regeneration of naive T lymphocytes and thymic function appeared by 6 months post transplant among the allogeneic recipients.26, 29
The recovery of the naive CD4+ T lymphocytes post HSCT has been shown to be protracted also in children.30, 31 In previous studies, data on immune reconstitution has been compared with either healthy, age-matched controls 31 or within subgroups. We used age-matched, immunosuppressed, non-transplanted controls to monitor the effects of previous chemotherapy and conditioning. Our study demonstrated that the recovery of naive T lymphocytes and TRECs was slow with one half of the recipients reaching the values of controls as late as 18 months post HSCT.
aGVHD has little effect on T cell regeneration.32, 33 Also in our study, aGVHD did not affect the regeneration of the naive T cells or TRECs post HSCT. However, and as previously reported,14, 15, 16, 29 low TREC levels did associate with extensive cGVHD post HSCT. Patients with cGVHD have a reduced capacity to produce naive CD4+ T cells34 because of thymic epithelial injury by GVHD and immunosuppressive drugs.
Our data is in agreement with previous studies showing an inverse correlation between the age of the recipient and regeneration of naive CD4+ T lymphocytes.2, 16, 18, 32 In addition, we also observed a negative correlation between thymic recovery and the age of the recipient, which is consistent with a damaging effect by GVHD on thymic function among the older recipients of stem cell grafts.7, 17 Thymic dysfunction on its part delays immune reconstitution and increases the for example incidence of infections early on post transplant.14, 15, 19
The recipients of bone marrow grafts receive a high amount of mature lymphocytes, which has been linked to lower incidence of infections.35 Our data on pediatric patients demonstrates that early herpesvirus infection(s) do not associate with a low level of naive T cells and TRECs, but associate with an increased amount of memory/active T cells. Furthermore, early herpesvirus infections post HSCT seem to identify a patient cohort with an adverse outcome poststem cell transplant. Early thymic recovery combined with an adequate amount and sufficient function of regulatory T cells appears to have a positive impact on transplant outcome.19 In our pediatric population, Foxp3 was used as a marker of CD25+CD4+ regulatory T cells having an important role in maintaining immunological self-tolerance. Foxp3 expression by T cells decreased during the first 3 months post transplant in all our three study groups and associated with a severe aGVHD at 3 months post HSCT.
Our study demonstrates the key effect of recuperation of the T cell compartment on a variety of factors influencing the outcome in pediatric stem cell transplantation. Importantly, the impact of therapy administered before transplant may have to be considered in for example designing the conditioning regimen, pre-emptive therapy of viral infections post transplant etc. The apparently more subdued immunological recovery among recipients of unrelated grafts may also affect our clinical thinking in the treatment of post transplant complications among these patients post HSCT. For post-transplant follow-up of T cell reconstitution flow cytometry appears more readily employable, but studies on the potential use of a pre- and/or post transplant ‘immunological profile’ in tailoring the therapy of an individual patient post transplant are warranted.
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We thank Inna Sareneva and Noora Alakulppi MSc for technical assistance. This study was supported by the Nona and Kullervo Väre Foundation and the Finnish Cultural Foundation and the Sigrid Juselius Foundation.
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Olkinuora, H., Talvensaari, K., Kaartinen, T. et al. T cell regeneration in pediatric allogeneic stem cell transplantation. Bone Marrow Transplant 39, 149–156 (2007). https://doi.org/10.1038/sj.bmt.1705557
- T cell receptor excision circle
- viral infection
- hematopoietic stem cell transplantation
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