Abstract
Our previous study showed that children who had been partially or completely thymectomized during heart surgery as infants had lower proportions and numbers of total lymphocytes and reduced proportions of T cells (CD3+), helper T cells (CD4+) and naive T cells (CD3+ CD4+ CD45RA+), but normal proportion of cytotoxic T cells (CD8+). In this study T lymphocytes from a selected group of eight of these children and age- and gender-matched controls were characterized further using flow cytometry to determine phenotypes of T cells and T cell subsets related to T cell regulation and phenotypes suggestive of extrathymic maturation. Immune function was assessed by measuring autoantibodies and antibodies against vaccines. The study group had significantly lower numbers of all the main subsets of T lymphocytes and the composition was different. Thus, the proportions of lymphocytes with the following phenotypes: CD3+, CD2+, CD7+, CD4+, CD62L+, CD4+ CD62L+ and CD4+ CD69− were significantly reduced in the study group compared with the control group, but significantly higher proportions were seen of lymphocytes expressing CD8α+ CD8β− and TCRγδ+ CD8α+ CD8β−. The absolute number and proportion of CD4+ CD25+ cells were reduced but the proportions of the subgroup of naive regulatory T cells (CD4+ CD25+ CD62L+) and non-activated regulatory T cells (CD4+ CD25+ CD69−) were not reduced in the thymectomized children. We conclude that the phenotypic characteristics of T lymphocytes of children who have lost their thymus in infancy are indicative of extrathymic maturation. T regulatory cells appear to be less affected than other subsets by the general reduction in T cell numbers.
Keywords: extrathymic T cell maturation, infants, T cells, T regulatory cells, thymectomy
Introduction
The precursors of T lymphocytes are generated in the bone marrow, except during the early fetal stage when they are generated in liver and spleen. From there they migrate to the thymus, which they enter as immature thymocytes. Within the thymus they undergo a series of maturation events, including the T cell receptor (TCR) gene rearrangement where the majority of T lymphocytes express TCR made up of αβ polypeptide chains, but only few express TCR composed of γδ chains [1,2]. Only a small proportion of thymocytes that mature in the thymus leave the thymus as naive T cells to circulate throughout the body ready to bind antigens that their TCRs recognize. Upon TCR engagement the T cells are activated, which leads to proliferation and differentiation of the T cells, which then become effector cells. Naive T cells express the homing receptor CD62L. Shortly after activation this receptor is down-regulated and activated T cells express the early activation marker CD69 as well as CD25, the interleukin (IL)-2 receptor α chain [1].
A small proportion of CD4+ T cells leaving the thymus constitutively express CD25. These CD4+ CD25+ T lymphocytes include T regulatory cells. They also express the CD152 antigen (CTLA-4), which is partly responsible for mediating their suppressive effects. CD4+ CD25+ T regulatory cells are thought to be involved in preventing autoimmunity and depletion of these cells in mice can result in development of autoimmune disease [3]. CD8+ CD28− T suppressor cells are another type of T cells that control immune responses and are important in the development of systemic tolerance [4]. They do not express the CD152 antigen, but inhibit proliferation of T cells by blocking activation of antigen-presenting cells [5]. Although the majority of T lymphocytes mature in the thymus, there are reports indicating that T lymphocytes may also differentiate outside the thymus [6,7]. It has been suggested that extrathymic maturation takes place in the intestines and in the liver [6–9]. The phenotype of these extrathymic T cells is in part different from that of T lymphocytes matured in the thymus. Their TCRs are also composed of either αβ or γδ polypeptide chains but they have a higher proportion of γδ T lymphocytes. Most of them are CD8+, but instead of expressing both the alpha and the beta chain of the CD8 molecule, the CD8 is an αα homodimer [6]. Some extrathymically derived T cells express the beta chain of the IL-2 receptor, but not the alpha chain (CD122+ and CD25−). These cells have been found to be generated in the liver [10]. Another subset of extrathymically derived T cells express neither the CD4 nor the CD8 co-receptors (CD3+ CD4− CD8−) [4]. Finally, T lymphocytes that express CD7 but not CD3 or the TCR (CD2+ CD3− CD7+) have been observed in the epithelium of the small intestine [11,12].
Our previous study showed that children who had undergone thymectomy as infants had a lower proportion and total numbers of T lymphocytes [13]. The aim of the current study was to perform a more detailed phenotypic analysis of T lymphocytes from these children and to compare them with lymphocytes obtained from healthy children. As the previous study had indicated low numbers of CD4+ T cells, these were analysed in more detail by testing for CD4+ CD25+, using CD69 and CD62L to distinguish between activated T cells and regulatory T cells. The presence of activated T regulatory cells with the phenotype CD4+ CD152+ was analysed as well as the T suppressor cell type with the phenotype CD8+ CD28−. Because depletion of CD4+ CD25+ cells can result in autoimmune disease, organ-specific autoantibodies were measured against thyroglobulin, parietal cells and pancreatic islet cells. The following markers were used to detect T cells displaying characteristics of extrathymically matured T cells: TCRγδ+ CD8αα+ and CD3+ CD4− CD8− T cells (intestinal), CD3+ CD25− CD122+ T cells (derived from the liver) and CD7+ CD3− T cells (intraepithelial). To test whether the thymectomy had affected immune responsiveness, IgG antibodies against measles, mumps and tetanus toxoid were measured, as all the children had received the relevant vaccines as infants.
Patients and methods
Subjects
The study group consisted of eight children, all of whom had participated in our previous study [13], and were selected from the original group because they had shown significant differences in numbers and proportions of T cells and T cell subsets. The children were 7–16 years of age (average age 12·1 years) and had undergone operation for congenital heart disease as infants (average age 2·5 months). Six children had a ventricular septal defect, one had transposition of the great vessels and one child had an anomalous pulmonary venous connection. As the thymectomy was not the reason for the operation, information about how much or if any part of the thymus had been removed was not always included in the surgical report. No information was available about thymectomy in three children, for two partial removal was recorded and in three all of the thymus had been removed. Eight healthy control subjects were matched for age and gender to the study group (average age 12·4 years). Written consent was obtained from all parents and the study was conducted with permission from the Icelandic National Bioethics Committee (VSN 01–142-V2-S1) and the Privacy and Data Protection Authority.
Data and sample collection
All the children in the study group and four children in the control group had already answered a detailed questionnaire in our previous study and were therefore asked only about changes in health from the previous study. The remaining four children in the control group were asked about history of infections, asthma, allergy, psoriasis, arthritis and antibiotic use. A peripheral venous blood sample was collected from all subjects.
Tests of immune system function
Haematological studies
White blood cell differential count and full blood count was performed at the Department of Haematology, Landspitali University Hospital using standard procedures (Coulter Counter analysis).
Autoantibodies
Autoantibodies against thyroglobulin, parietal cells and pancreatic islet cells were measured only in the study group, and as these were negative, sera from the control group were not tested. The measurements were carried out at the Department of Immunology, Landspitali University Hospital, using their standard methods (FEIA for antibodies against thyroglobulin and indirect immunofluorescence for antibodies against parietal and pancreatic islet cells).
Antibodies
The levels of IgG antibodies against tetanus toxoid, measles and mumps were measured by a solid phase enzyme-linked immunosorbent assay (ELISA) at the Departments of Immunology and Virology, Landspitali University Hospital.
Flow cytometry
Phenotypes of lymphocytes were examined by performing three-colour flow cytometric analyses using a fluorescence activated cell sorter (FACScan) flow cytometer (BD Biosciences, San Jose, CA, USA). Lymphocytes were stained with mouse anti-human monoclonal antibodies (mAbs) against the following surface antigens: CD2, CD3, CD4, CD7, CD8α, CD8αβ, CD25, CD28, CD62L, CD69, CD122, CD152 and TCRγδ using fluorescein isothiocyanate (FITC), phycoerythrin (PE) and peridinin chlorophyll (PerCP) or cyclin H (CyCh) as the fluorescent conjugates. Table 1 shows the combination of antibodies used and the phenotypes identified. In each tube 50 µl ethylenediamine tetraacetic acid (EDTA) anti-coagulated whole blood were incubated with 5 µl of each mAb on ice for 30 min. Red cells were lysed with 1000 µl lysis buffer (BD Biosciences), samples centrifuged for 5 min and then washed with 1000 µl of phosphate buffered saline (PBS). The tubes were centrifuged for 5 min, the supernatant discarded and the cell pellet resuspended and fixed with 400 µl of 0·5% paraformaldehyde. For each sample, forward and side angle light scatter profiles were used to acquire data for 10 000 events representing viable lymphocytes. Data were analysed with the CellQuest program (BD Biosciences). The data were expressed as proportion of positive cells (compared to cells stained with an irrelevant control antibody) or total number of positive cells. The mean and standard deviation (s.d.) was calculated for the study and control groups.
Table 1.
Combination of surface antigens analysed by flow cytometry and the phenotypes they identify.
| Tube no. | FITC | PE | PerCP/CyCh. | Phenotype defined |
|---|---|---|---|---|
| 1 | IgG2a | IgG1 | IgG1 | |
| 2 | CD4 | CD25 | CD62L | CD4+ CD25+ CD62L+: T regulatory cells |
| 3 | CD4 | CD25 | CD69 | CD4+ CD25+ CD69-: T regulatory cells |
| 4 | CD28 | CD8 | CD8+ CD28−: T suppressor cells | |
| 5 | CD4 | CD152 | CD4+ CD152+: activated T regulatory cells | |
| 6 | CD4 | CD3 | CD8 | CD3+ CD4-CD8−: extrathymically derived T cells, mainly found in intestinal mucosa |
| 7 | TCR γδ | CD8αβ | CD8α | TCR γδ+ CD8αα+: extrathymically derived T cells, mainly found in intestinal mucosa |
| 8 | TCR γδ | CD122 | CD25 | CD25− CD122+: T cells reported to develop in mouse liver |
| 9 | CD7 | CD2 | CD3 | CD3− CD7+: extrathymically derived T cells, mainly found in intestinal mucosa |
Statistical analysis
Student's t-test was used to compare differences between the mean values of the study and control groups, using Microsoft Excel®. A P-value ≤ 0·05 was considered statistically significant.
Results
Haematological parameters
Table 2 shows the results of routine haematological tests. The study group had lower numbers of lymphocytes (P < 0·001), but no other parameters analysed differed between the two groups. All values were within the normal range.
Table 2.
Comparison of blood status between study and control groups.
| Cell type | Study group × 109/l | Control group × 109/l | P-value |
|---|---|---|---|
| White blood cells | 4·76 ± 1·48 | 5·45 ± 0·80 | 0·27 |
| Platelets | 278·75 ± 70·69 | 325·50 ± 76·10 | 0·22 |
| Neutrophils | 2·59 ± 1·24 | 2·30 ± 0·51 | 0·56 |
| Lymphocytes | 1·56 ± 0·24 | 2·58 ± 0·46 | < 0·001 |
| Monocytes | 0·40 ± 0·12 | 0·43 ± 0·09 | 0·64 |
| Eosinophils | 0·18 ± 0·09 | 0·16 ± 0·16 | 0·85 |
| Basophils | 0·01 ± 0·04 | 0·01 ± 0·04 | 1·00 |
| Red blood cells ( × 1012/l) | 4·66 ± 0·36 | 4·87 ± 0·33 | 0·24 |
| Haemoglobin (g/l) | 138·38 ± 5·34 | 138·75 ± 9·45 | 0·92 |
| Haematocrit (l/l) | 0·41 ± 0·02 | 0·41 ± 76·10 | 0·22 |
All data given as mean ± s.d.
Autoantibodies
None of the children in the study group had measurable autoantibodies against parietal cells or pancreatic islet cells. One child in the study group had a marginally raised level for autoantibody against thyroglobulin or 350 IU/ml (normal level < 344 IU/ml) and all the remaining samples from the study group were negative.
Antibodies against tetanus toxoid, measles and mumps
No significant difference was found between the study and the control groups in levels of IgG antibodies against tetanus toxoid, measles and mumps (data not shown).
T cell phenotype analysed by flow cytometry
Tables 3 and 4 show the results of the flow cytometric analyses of lymphocyte phenotypes. Table 3 shows the data expressed as proportions of positive cells, whereas Table 4 shows the data expressed as the total numbers of lymphocytes expressing each phenotype. The study group had significantly lower proportions of lymphocytes expressing the following phenotypes: CD3+, CD2+, CD7+, CD4+, CD62L+, CD4+ CD62L+ and CD4+ CD69− compared with the control group. The study group had significantly higher proportions of lymphocytes expressing the phenotypes CD8α+ CD8β− and TCRγδ+ CD8α+ CD8β−.
Table 3.
Proportion of lymphocytes expressing an immunophenotype.
| CD antigen | Study group % | Control group % | P-value |
|---|---|---|---|
| CD3+ | 62·93 ± 10·63 | 73·77 ± 6·22 | 0·03 |
| CD2+ | 70·37 ± 6·95 | 80·26 ± 6·07 | < 0·01 |
| CD7+ | 46·17 ± 15·80 | 67·93 ± 12·95 | < 0·01 |
| CD3+ CD4– CD8− | 6·00 ± 2·08 | 4·80 ± 1·77 | 0·24 |
| CD2+ CD3− CD7+ | 8·71 ± 5·80 | 5·87 ± 2·94 | 0·24 |
| CD4+ | 35·23 ± 9·31 | 45·00 ± 5·81 | 0·03 |
| CD62L+ | 71·98 ± 5·14 | 79·88 ± 1·59 | 0·003 |
| CD4+ CD62L+ | 28·49 ± 8·93 | 40·80 ± 4·93 | 0·01 |
| CD4+ CD69+ | 0·15 ± 0·07 | 0·15 ± 0·11 | 0·85 |
| CD4+ CD69− | 35·58 ± 8·98 | 45·11 ± 5·61 | 0·03 |
| CD4+ CD25+ | 9·12 ± 2·50 | 9·67 ± 2·54 | 0·67 |
| CD4+ CD25+ CD62L+ | 7·22 ± 2·12 | 8·31 ± 2·76 | 0·39 |
| CD4+ CD25+ CD69− | 9·62 ± 2·78 | 9·29 ± 2·08 | 0·79 |
| CD4+ CD152+ | 2·11 ± 2·26 | 6·91 ± 11·74 | 0·29 |
| CD25− CD122+ | 33·33 ± 8·64 | 27·43 ± 4·06 | 0·11 |
| CD8α+ CD8β+ | 20·39 ± 5·12 | 23·45 ± 4·34 | 0·22 |
| CD8α+ CD8β− | 3·87 ± 1·95 | 1·90 ± 1·05 | 0·03 |
| CD8+ CD28− | 29 ± 5·01 | 25·97 ± 3·51 | 0·12 |
| TCRγδ+ | 12·26 ± 2·46 | 15·63 ± 6·68 | 0·21 |
| TCRγδ+ CD84 | 10·02 ± 2·12 | 10·34 ± 5·23 | 0·88 |
| TCRγδ+ CD8+ | 1·20 ± 0·98 | 4·05 ± 4·30 | 0·11 |
| TCRγδ+ CD8α+ CD8β− | 0·89 ± 0·30 | 0·33 ± 0·42 | < 0·01 |
All data given as mean ± s.d.
Table 4.
Number of lymphocytes expressing an immunophenotype.
| CD antigen | Study group × 109/l | Control group × 109/l | P-value |
|---|---|---|---|
| CD3+ | 0·99 ± 0·26 | 1·89 ± 0·33 | < 0·001 |
| CD2+ | 1·11 ± 0·27 | 2·06 ± 0·34 | < 0·001 |
| CD7+ | 0·73 ± 0·30 | 1·73 ± 0·36 | < 0·001 |
| CD3+ CD4− CD8− | 0·09 ± 0·03 | 0·12 ± 0·04 | 0·16 |
| CD2+ CD3− CD7+ | 0·14 ± 0·09 | 0·15 ± 0·07 | 0·78 |
| CD4+ | 0·55 ± 0·17 | 1·15 ± 0·23 | < 0·001 |
| CD62L+ | 1·12 ± 0·16 | 2·05 ± 0·35 | < 0·001 |
| CD4+ CD62L+ | 0·45 ± 0·16 | 1·05 ± 0·19 | < 0·001 |
| CD4+ CD69+ | 0·002 ± 0·001 | 0·004 ± 0·004 | 0·23 |
| CD4+ CD69- | 0·56 ± 0·17 | 1·16 ± 0·22 | < 0·001 |
| CD4+ CD25+ | 0·14 ± 0·04 | 0·26 ± 0·11 | 0·03 |
| CD4+ CD25+ CD62L+ | 0·11 ± 0·03 | 0·22 ± 0·12 | 0·03 |
| CD4+ CD25+ CD69− | 0·15 ± 0·04 | 0·24 ± 0·06 | 0·01 |
| CD4+ CD152+ | 0·03 ± 0·03 | 0·20 ± 0·40 | 0·26 |
| CD25- CD122+ | 0·52 ± 0·15 | 0·71 ± 0·20 | 0·06 |
| CD8α+ CD8β+ | 0·32 ± 0·12 | 0·61 ± 0·17 | 0·002 |
| CD8α+ CD8β– | 0·06 ± 0·03 | 0·05 ± 0·03 | 0·48 |
| CD8+ CD28− | 0·47 ± 0·14 | 0·67 ± 0·12 | 0·01 |
| TCRγδ+ | 0·19 ± 0·05 | 0·39 ± 0·15 | 0·01 |
| TCRγδ+ CD8− | 0·16 ± 0·04 | 0·39 ± 0·10 | 0·03 |
| TCRγδ+ CD8+ | 0·02 ± 0·02 | 0·11 ± 0·12 | 0·08 |
| TCRγδ+ CD8α+ CD8β− | 0·014 ± 0·005 | 0·009 ± 0·011 | 0·27 |
All data given as mean ± s.d.
Table 4 shows the numbers of lymphocytes expressing the phenotypes analysed. The numbers of T lymphocytes were reduced significantly in the study group, as expected, and this was reflected in all T cell subsets, including those that showed equal or increased proportions (Table 3). This indicates a significant difference in the composition of the peripheral T cell pool in the thymectomized children.
In summary, the children in the study group had significantly lower numbers and proportions of total T lymphocytes. They had lower numbers and proportions of T helper cells (CD4+), naive T cells (CD4+ CD62L+) and non-activated T cells (CD4+ CD69−). Notably, this difference was not reflected in T regulatory cells, where the study group had a higher proportion of non-activated T regulatory cells (CD4+ CD25+ CD69−). No difference was found in the proportion of cytotoxic T cells (CD8+, expressing both CD8α and CD8β); however, the study group had a higher proportion of cytotoxic T cells with CD8αα as well as TCRγδ T cells expressing CD8αα.
Clinical data
The incidence of allergy, psoriasis or otitis media since the time of the previous study was six of eight children in the study group and two of eight children in the control group. None of the participants during this time had had asthma, meningitis or other infections, nor had they been hospitalized.
Discussion
The purpose of this study was to examine the phenotype and function of T lymphocytes in children who have lost all or part of their thymus as a result of heart surgery in early infancy. As expected from our previous study and other published reports, the study group had a lower total number of lymphocytes as well as lower numbers of T cells (CD3+) and helper T cells (CD4+) [14–16]. These results indicate that T cell maturation continues even though the thymus has been lost, but it is not as efficient as when the intact thymus is present. In the current study we have shown that the T lymphocytes of these children were also phenotypically different from those of age-matched controls. Thus, the study group had lower proportions and total numbers of naive lymphocytes (CD62L+) and naive T cells (CD4+ CD62L+) as well as non-activated T cells (CD4+ CD69−), which include both naive and memory T cells. The proportions of the subgroups of naive and non-activated regulatory T cells (CD4+ CD25+ CD62L+ and CD4+ CD25+ CD69−) were, however, not reduced in the thymectomized children. The lower total lymphocyte numbers in the study group were also reflected in the reduced numbers of cytotoxic T cells (CD8αβ+) and T suppressor cells (CD8+ CD28−) but not in their proportion. The study group had higher proportions of CD8αα+ T cells and TCRγδ+ CD8αα+ T cells, which is consistent with extrathymic maturation. The numbers of cells with these phenotypes were not reduced, in contrast to other CD8+ T cell subsets. Thus, extrathymic T cells and T regulatory cells were less affected than other T cell subsets by the overall reduction in T cell numbers following thymectomy. Table 5 shows a comparison of published phenotypes for extrathymically matured T cells with our findings.
Table 5.
Data presented in this study related to published T cell phenotypes that distinguish T cells that originate from thymic or extrathymic maturation.
| Phenotype | Thymic maturation | Extrathymic maturation | Data in this study |
|---|---|---|---|
| CD4+ | + + | + | ↓ |
| CD8αβ+ | + + | + | = |
| CD8αα+ | − | + | ↑ |
| CD3+ CD4−CD8− | − | + | ↑ n.s. |
| TCRαβ+ | + + | + | Not measured |
| TCRγδ+ | + | + + | = |
| TCRγδ+ CD8αα+ | + | + + | ↑ |
| CD25− CD122+ | − | + | ↑ n.s. |
The information regarding thymic and extrathymic maturation is based on [6,7,10,16–18], indicating whether the subsets are present in high (+ +) or low (+) proportions or absent (−). Arrows indicate whether the proportions were increased or decreased in the study group compared with the control group. No difference is shown by an equals sign; n.s.: not significantly different from control group.
T cell maturation outside the thymus has been studied mainly in mice where the intestines and the liver have been identified as the major maturation sites [6,9,10,17]. Wang et al. studied the maturation of human T cells by transplanting human haematopoietic stem cells into congenitally athymic mice [18]. The phenotype of T cells that matured in these mice was compared with that of T cells from postnatal thymus, bone marrow and umbilical cord blood and peripheral blood leucocytes. The T cell pool that matured extrathymically had higher numbers of cells that expressed the surface antigen CD8 made up of αα-chains rather than αβ-chains. These results agree with our data showing a significantly higher proportion of CD8αα+ T lymphocytes in the study group as well as a higher proportion of CD8αα+ T cells that also express TCRγδ. It has been shown previously in mice that T cells maturing outside the thymus have a higher proportion of cells that express the γδ receptor rather than the αβ form, which is thought to reflect less efficient positive selection in the gut compared with the thymus [17]. In the study by Wang et al., double-negative T cells (CD3+ CD4− CD8−) were more prominent after extrathymic maturation compared with thymic maturation [18]. Although not statistically significant, the study group had a higher proportion of CD3+ CD4− CD8−.
In our previous study the thymectomized children had lower numbers of naive T cells of the CD3+ CD45RA+ phenotype. This finding was supported further in the current study, showing a reduction in naive T cells of the CD4+ CD62L+ phenotype and non-activated T cells (CD4+ CD69−) in the study group. The study by Wang et al. showed that T cells that matured in athymic mice contained a lower proportion of cells expressing CD62L [18]. The pool of naive and non-activated T cells is thus reduced following thymectomy.
Two T cell phenotypes that have been associated with maturation in the liver (CD25− CD122+) [9] or identified in the epithelium of small intestine (CD2+ CD3− CD7+) [11,12] were not altered in proportion or total numbers in our study group. The rate of maturation of these cells would therefore appear not to depend on the presence of the thymus.
In normal adult life the T cell pool is maintained by peripheral homeostatic T cell proliferation. This mechanism also contributes substantially to T cell expansion and regeneration following depletion (e.g. by chemotherapy) [19,20]. In euthymic children all T cell subsets recover. In children with reduced thymic function, as well as in adults, CD4+ T cells show poor recovery whereas CD8+ T cells regenerate well. Interestingly, under these conditions the composition of the CD8+ subset has been shown to be altered, with a predominance of CD8+ CD28– and CD8+ CD57+ cells.
Even though the thymectomized children had a lower proportion of helper T cells (CD4+), the proportion of T regulatory cells (CD4+ CD25+) was not affected in the study group. One possible explanation for the retained frequency of T regulatory cells would be that these cells mature earlier in life than other CD4+ cells and that they were therefore less affected by the thymectomy.
Both the current study and our previous study indicated that loss of the thymus in infancy had not had clinical consequences or affected the immune function of the children in terms of frequency of infections, response to childhood vaccinations or presence of organ-specific autoantibodies. It remains to be seen whether this continues into adulthood, as it might be expected that less effective negative selection during extrathymic maturation could lead to increased survival of autoreactive cells.
Very few reports have been published on the impact of thymectomy in human infants. These reports have documented lower numbers of T helper cells (CD4+) [14–16] and no important clinical effects on the immune system have been detected. Two studies found evidence of decreased numbers of CD8+ cells [14,16], which is in accordance with our data. However, the frequency of CD8+ cells was not affected in the study group. Halnon et al. concluded that they found no evidence of extrathymic T cell generation based on the finding that TRECs (T cell receptor recombination excision circles) were undetectable in many of their subjects that had no residual thymus [16]. TREC is a non-replicating circular DNA that is generated during excisional rearrangements of the coding sequences for T cell receptor proteins. It has been used to detect recent thymic emigrants and quantify thymic output, but recent reports reveal that these data should be interpreted with caution [21]. Another marker, CD103, has been proposed as a marker for recent thymic emigrants [22]. In our previous study, the numbers of T cells positive for this marker were increased in the thymectomized children [13]. Because CD103 is also expressed on T cells in or destined for the mucosa [23], the increase observed previously may be an indication of an increase in T cells linked with the gut mucosa.
In conclusion, this study has shown evidence for extrathymic T cell maturation in children who underwent heart surgery and partial or total thymectomy as infants, but the site of maturation is unknown. Compared with age-matched control children, the T cells of these children had significantly different phenotypes that were consistent with phenotypes described for extrathymically matured T cells in animal studies. No clinical consequences were detected so far but it remains to been seen whether these might occur later in life.
Acknowledgments
The authors wish to thank Dr Ingileif Jonsdottir and Mrs Gunnhildur Ingolfsdottir at the Department of Immunology, Landspitali University Hospital for their help with measuring antibodies against tetanus toxoid and Dr Thorgerdur Arnadottir and Mr Asgeir E Asgeirsson at the Department of Virology, Landspitali University Hospital for their help with measuring antibodies against measles and mumps.
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