Longitudinal analysis of T cells responding to tetanus toxoid in healthy subjects as well as in pediatric patients after bone marrow transplantation: the identification of identical TCR–CDR3 regions in time suggests long-term stability of at least part of the antigen-specific TCR repertoire

Barbara C. Godthelp, Maarten J. D. van Tol, Jaak M. Vossen and Peter J. van den Elsen

Departments of Pediatrics and Immunohematology and Blood Transfusion, Leiden University Medical Center, Building 1, E3-Q, PO Box 9600, 2300 RC Leiden, The Netherlands

Correspondence to: P. J. van den Elsen


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
To understand the nature of long-term Th immune responses, we investigated in the present study the TCRBV gene repertoire of CD4+ T cells specific for the recall antigen tetanus toxoid (TT) in recipients of an allogeneic bone marrow transplantation (allo-BMT) at several time points after transplantation and in their BM donors. We observed that the TCR repertoire of TT-specific CD4+ Th cells was heterogeneous, and differed between allo-BMT recipients and their respective donors. Some individuals, however, used similar TCR–complementarity-determining region (CDR) 3 motifs that could reflect recognition of and selection by similar promiscuous epitopes of TT. Longitudinal analysis of this TT-specific T cell response revealed that T cells with completely identical TCR were present at several time points after the first analysis in allo-BMT recipients, most probably reflecting long-term stability of at least part of the antigen-specific TCR repertoire. Similar stability of the TT-specific TCR repertoire in time was also noted in the allo-BMT donors. These observations reveal that within a given individual the dominant antigen-specific T cell clones persist in time in an otherwise diverse TT-specific CD4+ T cell immune response.

Keywords: clonal expansion, human, repertoire development, T lymphocytes, TCR


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Tetanus, caused by the bacterium Clostridium tetani, is a quite rare disease among humans, but is still a major health problem in underdeveloped countries (13). Tetanus-related mortality is due to the secreted tetanus toxin (TTX) that blocks the release of neurotransmitters (4,5). Tetanus can be easily prevented by vaccination procedures with a partially denatured non-toxic form of TTX, tetanus toxoid (TT) (6). However, protective immunity against tetanus declines with increasing age (2,7) and these older individuals therefore have an increased risk of developing tetanus. Because the majority of the Western population has been vaccinated with TT, humoral and cellular responses against this T cell-dependent recall antigen are used to monitor the general immune status of individuals.

T cells recognize antigenic peptides that are present in the MHC class I or class II molecules on the antigen-presenting cell (APC), through the TCR. T cells of healthy individuals display TCR repertoires that are diverse as a result of recombination of different TCR gene segments, imprecise joining of the segments, and N- and P-nucleotide addition (8,9). However, as a result of non-random TCR gene segment usage, non-random amino acid incorporation in the complementarity-determining region (CDR) 3 and skewing of certain TCRBV/TCRAV gene segments towards either CD4+ or CD8+ T cell subsets (1014), the available peripheral repertoire is less diverse than theoretically possible. Recognition of MHC–peptide complexes by TCR occurs through interaction of hypervariable or CDR regions within the TCR V segment, of which CDR2 predominantly interacts with the MHC {alpha} helices, CDR1 interacts with both the MHC and the antigenic peptide, and CDR3 predominantly interacts with the antigenic peptide (1519).

The adaptive immune response to infectious agents is characterized by initial priming and expansion of complex pathogen-specific T cell populations. The elicited T cells participate in the host defense by controlling the infection and eradicating the pathogen either directly in the case of CD8+ cytotoxic T cells or indirectly via secretion of cytokines in the case of CD4+ Th cells. Survival of these effector T cells in the periphery depends on continuous TCR–MHC contact (20,21). In general, the primary antigen-specific CD4+ and CD8+ T cell responses in humans are heterogeneous, even when defined antigenic peptides were used (2224), although some sharing of TCR gene segments has been reported (25,26). The long-term immune responses, in particular the Th cell responses, are less well characterized. Evidence from mouse models suggests that long-term immune responses are mediated by a limited number of T cell clones (27,28) and in humans some evidence on long-term preservation of an (heterogeneous) effector CD8+ cytotoxic T lymphocyte response has been reported (2931).

After allogeneic bone marrow transplantation (allo-BMT), two mechanisms of T cell reconstitution can be distinguished. First, peripheral expansion of graft-derived mature donor T cells which provides the first wave of T cells after allo-BMT (3234). These T cells have restricted TCR repertoires (35) and can be maintained in the periphery for >10 years (36). The second mechanism involves selection of BM-derived precursor T cells of the donor either in the thymus (3739) or extrathymically (40,41). The latter process, in which these donor-derived precursor T cells are educated in the thymus through interactions with complexes of allo-MHC and peptide of the recipient, most probably accounts for the more durable reconstitution of the T cell immune repertoire (42). Since this donor-derived T cell immune repertoire is also shaped on allo-MHC molecules of the recipient, it may differ in composition from the original donor T cell immune repertoire.

In this study we investigated the TCR ß chain repertoire of CD4+ T cells specific for the recall antigen TT in allo-BMT recipients at several time points after transplantation. In order to evaluate the development of the TT antigen-specific T cell repertoire and to gain more insight into the preferred pathway of T cell immune reconstitution after allo-BMT. We observed that the TCR repertoire of TT-specific CD4+ T cells was heterogeneous with a general lack of similarity of TCR gene segment usage between allo-BMT recipients and their donors which reflects education of the donor-derived T cell precursors on recipient-derived allo-MHC molecules. Furthermore, the TT-specific T cell repertoire was found to be stable in time, i.e. T cells with completely identical TCR gene segments could be identified for several consecutive years after the first analysis, in allo-BMT recipients as well as in healthy donors. In addition, similar TCR–CDR3 motifs were observed in the TCR ß chain variable regions of TT-specific T cells derived from several unrelated individuals that reflecting recognition of shared TT epitopes.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Patient/donor selection and mononuclear cell collection
We selected six allo-BMT recipients of which material of the donor was available. Two of these allo-BMT recipients were treated for leukemia and four for (severe) combined immunodeficiency diseases [(S)CID]. The characteristics of all analyzed individuals and their HLA typing are summarized in Tables 1 and 2GoGo respectively. All allo-BMT recipients were included in a re-vaccination protocol with diphtheria toxoid (D)–tetanus toxoid (T)–inactivated poliovirus type I, II and III (IPV) at 10, 14 and 24 weeks after allo-BMT. Blood was drawn from all allo-BMT recipients at two or more time points after transplantation (with the exception of UPN 275) and from their donors at one or two time points (Table 1Go). The first blood sample of the allo-BMT recipients was taken at least 1 year after transplantation since TT-specific immune responses cannot be detected earlier (44). Two of these recipients, UPN 53 and UPN 59, and their BM donors received an additional TT-booster vaccination 4 weeks prior to the collection of blood, i.e. 11 years after BMT. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-isopaque (LUMC Hospital Pharmacy, Leiden, The Netherlands) density gradients, and were either taken into culture or cryopreserved in RPMI with 20% human serum and DMSO at a final concentration of 10% until further use.


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Table 1. Characteristics of all analyzed individuals
 

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Table 2. HLA typing of BMT recipients and BM donors
 
Generation of TT-specific T cell lines
TT-specific T cell lines were generated and tested as described previously (42). Briefly, 3–6x106 PBMC were grown in culture medium in the presence of 1.9 limes flocculationis/ml TT (RIVM, Bilthoven, The Netherlands). After at least two rounds of TT re-stimulation, T cell clones were generated from some of these TT-specific T cell lines by limiting dilution (45). TT specificity of the T cell lines/clones was determined by a standard [3H]thymidine incorporation assay using TT-coated Epstein–Barr virus-transformed B lymphoblastoid cell lines (BLCL) of recipients and donors as APC. The T cell lines and clones that, after repeated testing, had a stimulation index (SI) > 3, with >1000 c.p.m., were considered to be TT specific (SI = 3H incorporation of T + APC + TT/3H incorporation T + APC only). All T cell clones analyzed in this study were tested for CD4 cell-surface expression by flow cytometry with a FACScan (Becton Dickinson, Mountain View, CA) using fluorescein-conjugated CD4 antibodies (Dakopatts, Glostrup, Denmark).

Fine specificity of the TT-specific T cell clones was determined by testing the proliferation of these clones against previously reported epitopes of TT (46,47). The amino acid sequences of the peptides used are as follows: P2 (TT amino acids 830–844), QYIKANSKFIGITEL; P12 (TT amino acids 580–599), NSVDDALINSTKIYSYFPSV; P21 (TT amino acids 916–932), PGINGKAIHLVNNESSE. Peptides were synthesized by solid-phase strategies on an automated multiple peptide synthesizer (AMS 422; Abimed, Langenfeld, Germany). Proliferation assays with these peptides were performed as described above. Peptide was loaded onto the autologous BLCL, which were used as APC, by incubating BLCL with 5 µg/ml of peptide at 37°C for 1 h prior to testing.

RNA isolation, cDNA synthesis and PCR amplification
Total RNA was extracted from the TT-specific T cell clones (1x105–1x106 cells) and converted into cDNA using oligo(dT) (Promega, Madison, WI). The cDNA was subjected to PCR amplification for the determination of the TCRB and TCRA gene segment usage. PCR amplification and TCR primers were as described previously (14,42).

PCR fragment purification and DNA sequencing
The desired TCRAV or TCRBV PCR fragments were purified by electrophoresis in a 1% low-melting-point agarose gel and subsequent use of Wizard columns (Promega). The Wizard-purified fragment was used for direct sequencing (48) using the T7 sequencing Kit (Pharmacia, Uppsala, Sweden) with 5–10 pmol of TCRAC/BC internal primer, ~0.25 pmol PCR fragment and [{alpha}-33P]dATP (0.5 µCi) (Dupont NEN, Boston, MA). DNA sequences were compared with TCR sequences contained in the GenBank using the PCGENE computer software program (release 6.85, Intelligenetics, Palo Alto, CA). The primers that were used for sequencing of the TCR of TT-specific T cell clones were: TCRAC internal, 5'-GGT ACA CGG CAG GGT CAG GGT TC-3'; TCRBC internal, 5'-TGT GGG AGA TCT CTG CTT CTG-3'.

Longitudinal analysis of TCR repertoire: presence of identical TCR–CDR3 sequences in time
For analysis of persistence of TCR clonotypes, TCRBV PCR products were generated from TT-specific T cell lines and clones (42), size fractionated in a 1% agarose gel, and transferred to nylon membranes (Hybond-N+; Amersham, Little Chalfont, UK) in 20xSSPE for 17 h. After blotting, filters were incubated in 0.4 N NaOH for 20 min and washed in 5xSSPE for 5 min at room temperature. Filters were prehybridized and hybridized at 60°C in 5xSSPE, 0.1% BSA, 0.1% Ficoll, 0.1% polyvinylpyrolidone and 0.5% SDS. The oligonucleotide probes specific for the TCR–CDR3 region (see Table 8Go) were end-labeled with [{gamma}-32P]ATP as described previously (42). After the hybridization in an incubator (Appligene, Pleasanton, CA) for ~17 h at 60°C, the filters were washed once in 6xSSPE/0.1% SDS, pH 7.5 for 15 min at 60°C and once in 6xSSPE/0.1% SDS, pH 7.5 for 15 min at the Tm of the oligonucleotides. After washing, filters were exposed to X-ray films at –80°C for 2–4 h.


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Table 8. Nucleotide sequences of TCR–CDR3 oligonucleotide probes used for patients/donors
 

    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Generation of TT-specific T cell lines—an attempt to determine fine specificity
To study the kinetics of CD4+ T cell immune responses in time, TT-specific T cell lines were generated by stimulation of PBMC, collected at several different time points from 1 year after BMT onwards, with TT. The responding T cells showed specificity for TT in a [3H]thymidine incorporation assay with SI = 7–83 (Table 3Go) in allo-BMT recipients that is similar to their BM donors (SI = 9–128). Upon mitogenic stimulation with phytohemagglutinin, all allo-BMT recipient-derived T cell lines showed proliferative responses with SI = 43–412 (Table 3Go) that were in the same range as those of their BM donors (SI = 37–590). Subsequently, T cell clones were generated from the TT-specific T cell lines by limiting dilution and only those clones that exhibited TT specificity upon repeated testing were used for further molecular analysis. As can be seen from Table 3Go, following this procedure, between 6 and 59% of the tested clones were found to be TT specific. This dissimilarity in the percentage TT-specific T cells most likely corresponds to differences in precursor frequency of T cells responding to TT in the analyzed individuals. All generated TT-specific T cell clones were CD4+ as determined by FACS analysis and expressed the {alpha}ß TCR (results not shown).


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Table 3. Proliferative capacities of TT-specific T cell lines and percentage of TT-specific clones at several time points after transplantation
 
An attempt was made to determine the fine specificity of a selection of established TT-specific T cell clones/lines by testing the proliferation of these T cells clones against previously reported promiscuous TT epitopes P2 (TT amino acids 830–844), P12 (TT amino acids 580–599) and P21(TT amino acids 916–932) (45,46). P2/P12/P21-specific proliferation could not be observed in the TT-specific T cell clones (SI < 3) revealing a lack of recognition of these peptides in our panel of T cell clones (Table 4Go). However, reactivity to P2 and P21 could be observed in the TT-specific T cell line of D(UPN53) and of UPN293 showing that T cells able to recognize these peptides are present in the lines which were used to obtain the T cell clones.


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Table 4. Determination of fine-specificity of established TT-specific T cell clones
 
TCRBV gene usage and CDR3 analysis of TT-specific T cell clones
Using TCRBV PCR and DNA sequencing, we studied the TCR repertoires of the TT-specific T cell clones. This analysis showed a heterogeneous TCR repertoire in the majority of the allo-BMT recipients and in their donors (Table 5Go). At least one TCR sequence was found multiple times in each individual, revealing the dominance of these particular T cell clones in the respective TT-specific T cell line. These dominant TT-specific T cell clones were always accompanied by other T cell clones expressing different TCR (Table 5Go). This varied between one additional T cell clone in D(UPN59) to 12 additional T cell clones as observed in D(UPN293) and in allo-BMT recipient UPN285. The variation in the number of additional T cell clones may be related to the time period between this analysis and the last TT (booster) vaccination and/or with the above-mentioned TT precursor frequencies. When comparing allo-BMT recipient-derived sequences with those from their donors, striking dissimilarities were observed in TCRBV, TCRBJ usage and in TCR–CDR3 amino acid composition irrespective of the conditioning regimen (mild/myeloablative) and of the type of BM graft (T cell depleted/unmanipulated) (Table 5Go). T cell clones with an identical TCRB amino acid composition were observed in the donor-recipient combination, D(UPN235) and UPN235 (Table 5Go), albeit with a different TCRB nucleotide composition (results not shown) and TCRA chain (Table 6Go). This indicates that homologous but different TCR were used in the allo-BMT recipient and in the corresponding BM donor.


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Table 5. Results of TCR DNA sequence analysis in allo-BMT recipients at the first time point of analysis after BMT and of their BM donors
 

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Table 6. TCRB and TCRA usage of TCRBV5S6-expressing T cells with homologous TCR–CDR3 regions derived from several (unrelated) individuals
 
Detection of a conserved CDR3 motif in several different individuals
T cell clones expressing a TCRBV5S6+ T cell receptor with a very homologous TCR–CDR3 region with the motif XGLA(G)X(X) (Table 5Go) that were found in four (unrelated) individuals i.e. UPN235 and donor, D(UPN293) and D(UPN285), were also analyzed for their TCRA gene segment usage (Table 6Go). This TCR {alpha} chain analysis demonstrated that three of the six analyzed T cell clones used the TCRAV19S1 gene segment but differed in N region and in TCRAJ gene segments. Similarly, two clones shared usage of TCRAV1S7 but also differed in N region and in TCRAJ gene segments (Table 6Go). Furthermore, in D(UPN285) two in-frame rearrangements of the {alpha} chain, i.e. TCRAV14S1 (GPR) and TCRAV19S1 (RSS), were observed. The striking conservation of TCRBV sequences and the sharing of TCRAV sequences that was observed in these TT-specific T cell clones of unrelated individuals most probably reflect recognition of a similar promiscuous T cell epitope of TT. This is because the HLA-DR typing of these individuals was not identical, although three individuals shared the HLA-DR17 allele (Table 2Go and Table 7Go).


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Table 7. Presence of TCR clonotypes: evidence for long-term TCR repertoire stability
 
Longitudinal analysis of the TCR repertoire: evidence for long-term stability of the TCR repertoire of TT-specific T cells
In order to study the stability of the TCR repertoire of T cells responding to TT, we used oligonucleotide probes based on the most frequently found TCR sequences of each individual (Table 5Go) to screen the TT-specific T cell lines generated at several time points after the first analysis for the presence of these dominant TCR sequences. Examples of TCR–CDR3 oligonucleotide hybridizations are shown in Figs 1 and 2GoGo. The screening revealed the presence of T cells expressing the dominant TCR (Table 5Go) in six of the seven individuals who were analyzed with at least a 1-year interval (Table 7Go) which points to long-term stability of the dominant building blocks of the anti-TT TCR repertoire. The only exception was UPN 235 who lacked T cells expressing the TCRBV5S6+-QGLAGV or the TCRBV4S1+-VSTGAGTAA TCR at 4 years after allo-BMT (Table 7Go). Furthermore, in UPN53 and in UPN59 the presence of T cells expressing the dominant TCR sequence of the donor, TCR8S1/2 PDRGSP [D(UPN53)] and TCRBV13S1 TLGAPN [D(UPN59)] respectively, could be demonstrated at 11 years after allo-BMT (Fig. 1Go), upon hybridization of patient-derived T cell lines with donor TCR-specific oligonucleotide probes. The hybridization signal of these recipient-derived T cell lines was significantly lower than that of the donor- derived T cell lines, indicating that the amount of donor-type T cells in the recipient was very low but visible after PCR and subsequent hybridization with TCR–CDR3-specific oligonucleotide probes. Moreover, it should be noted that these T cells with a donor-type TCR were not found amongst the recipient-derived TT-specific T cell clones (Table 5Go). This suggests that the frequency of these donor T cells in the TT-specific T cell lines of both recipients was too low to give rise to donor-type T cell clones after limiting dilution. From UPN 285 we were able to analyze the TCR repertoire for 3 consecutive years after allo-BMT which also revealed repertoire stability. T cells with the dominant TCRBV1S1 and TCRBV9S1 TCR were found at all three time points, whereas T cells expressing the TCRBV5S6 TCR could only be detected at 1 and 3 years after allo-BMT (Table 7Go and Fig. 2Go). However, the presence of T cells with the TCR TCRBV5S6 GGLAGI that was dominant in the donor could not be demonstrated in UPN 285 (Table 7Go), suggesting that BM graft-derived memory T cells did not contribute to the anti-TT T cell response in this individual.



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Fig. 1. Longitudinal analysis of T cells responding to TT: evidence of TCR repertoire stability in anti-TT responses. Shown are the Southern blots of TCRBV2S1, TCRBV8S1/2 and TCRBV13S1 PCR products of TT-specific T cell lines and clones obtained from UPN53 and UPN 59 and their donors at several time points after allo-BMT using the TCR–CDR3-specific oligonucleotides (Table 7Go) as hybridization probes. L1, TT-T cell line 11 years after allo-BMT; C1, T cell clone at 11 years post-BMT; L2/C2, line/clone 14 years post-BMT [UPN59 and D(UPN53)] or 15 years post-BMT [UPN53 and D(UPN59)]; L3, line 15 years post-BMT; L rec., T cell line of recipient; N, negative control. The TCR–CDR3 sequence for which the oligonucleotide probe was designed is given in bold; Tm of these probes are also depicted.

 


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Fig. 2. TCR repertoire stability at 3 consecutive years after allo-BMT upon longitudinal analysis of T cells responding to TT. Southern blots of TCRBV1S1, 5S1 and 9S1 PCR products of UPN 285-derived TT-specific T cell lines and clones using the TCR–CDR3-specific TCRBV1S1 (GLG), 5S1 (YRGLAQG) and TCRBV9S1 (PTGSG) oligonucleotides (Table 7Go) as hybridization probes. L1, TT-T cell line 1 year after allo-BMT; C1, T cell clone at 1 year post-BMT with TCRBV family (TCRBV1, TCRBV5, TCRBV8, TCRBV9 and TCRBV 18); L2/C2, line/clone 2 years post-BMT; L3, line 3 years post-BMT. The TCR–CDR3 sequence for which the oligonucleotide probe was designed is given in bold; Tm of these probes are also depicted.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study we have evaluated the TCR ß chain repertoire of TT-specific CD4+ T cells in allo-BMT recipients at several different time points after transplantation and in their BM donors. TT-specific T cell lines could be generated from all analyzed individuals, albeit with different numbers of genuine TT-specific T cell clones which is suggestive of differences in precursor frequencies of T cells responding to TT. In general, the anti-TT precursor frequencies are relatively low (45), even in recently (re-)vaccinated individuals, and vary from 1 to 10 TT-reactive T cells in 1x106 PBMC. The T cell clones isolated from the TT-specific T cell lines that were not TT specific most probably represent T cells that benefit from the cytokine production of the genuine TT-specific T cell clones (52). We showed that in all individuals multiple TCR were used by the TT-specific T cells with variation amongst individuals reflecting differences in TCR repertoire shaping on individually expressed complexes of peptide and MHC class I or class II molecules. In particular, when comparing allo-BMT recipient-derived TCR sequences of the TT-specific clones with those from their donors, a striking dissimilarity was observed in TCR gene segment usage and in amino acid composition of the CDR3 irrespective of the conditioning regimen given prior to allo-BMT or the type of BM graft. This could infer that donor-derived T cell precursors are educated on complexes of allo-MHC and peptide of the recipient.

In each individual at least one TCR sequence was found multiple times, revealing the dominance of T cells expressing these TCR in the generated lines, which is in concordance with previously published data (22,24,26). This clonal dominance may be the result of the in vitro expansion period of the T cell line favoring T cells with a growth advantage. However, the possibility that multiple copies of the TT-specific T cells were present in peripheral blood, presumably as a result of repeated exposure to the antigenic compound, cannot be excluded. In theory, graft-versus-host disease and/or the immunosuppression given after BMT might also influence the shaping of the TCR repertoire. However, in our study no patients with serious graft-versus-host disease were included and all received immunosuppressive therapy during 6 months after BMT. All molecular analyses were therefore performed after the withdrawal of the immunosuppressive therapy.

The mechanisms that are involved in the complete restoration of the T cell immune repertoire after allo-BMT in humans are not completely understood. Selection of precursor T cells either in the thymus (3739) or extrathymically (40,41) as well as peripheral expansion of graft-derived mature donor T cells (3234) are thought to play important roles in this process. Moreover, it has been reported previously that BM grafts may contain T cells that are specific for microorganisms (5355) such as Mycobacterium Tuberculosis and the varicella zoster virus. These T cells may, therefore, play an important role in the host defense early after transplantation, especially in recipients of unmanipulated BM grafts. We were only able to identify T cells with a donor-type TCR in the SCID patients after BMT and only upon hybridization with radiolabeled TCR–CDR3 oligonucleotides. This suggests that the process of peripheral expansion plays a minor role at the time points we analyzed after allo-BMT or, alternatively, that the frequency of these donor-type T cells was below detection level in our patients. However, it cannot formally be excluded that these T cell clones were precursor T cells that were selected in the thymus of the recipient to recognize a similar MHC class II–peptide complex and, therefore, have identical TCR as the T cell clones isolated from the donor. The lack of similarity of TCR sequences between donors and recipients despite the observed repertoire stability within one individual is suggestive for a major role of precursor T cell selection in the complete reconstitution of the T cell immune repertoire (44).

Although the TCR repertoire of TT-specific T cell clones was found to be heterogeneous, four (unrelated) individuals used the TCRB CDR3 motif XGLA(G)X(X) which is suggestive for the recognition of and selection by a similar promiscuous epitope of TT because HLA typing of these individuals was not completely identical. However, fine specificity of these T cell clones to previously reported (promiscuous) immunodominant epitopes of TT P2 (amino acids 830–44), P12 (amino acids 580–99) and P21 (amino acids 916–32) (46,47) could not be demonstrated. Alternatively, determination of the fine-specificity using a complete set of overlapping peptides spanning the complete TT sequence may shed more light on the epitope recognized by these TT-specific T cell clones. Since three out of four individuals bearing TCR with this motif are HLA-DR17, searching for peptides with HLA-DR17-binding motifs in TT might be possible. However, finding the TT peptide which is recognized by the TCR bearing the XGLA(G)X(X) motif may be problematic because single TCR may have multiple specificities, i.e. recognize more than one epitope (5658).

Longitudinal studies of TCR repertoires derived from antigen-reactive T cells, i.e. long-term immune T cell responses are rare. There is evidence to suggest some repertoire stability in CD8+ cytolytic T cells such as those directed against the Epstein–Barr virus-derived nuclear antigen EBNA-3 (30), the HIV-derived glycoprotein 41 transmembrane protein (29), influenza A (31,59) and HTLV-1 (60). Because our understanding of the nature of CD4+ Th cell responses is even more limited (61,62), we have analyzed the TCR repertoires of TT-specific T cells in our panel of allo-BMT recipients at several different time points after BMT and of their BM donors. This analysis showed that the TCR repertoires remained heterogeneous at later time points and that the individual-specific building blocks of the long-term anti-TT T cell response were stable in time, i.e. TT-specific Th cells with completely identical TCR could be identified at several consecutive years after the first analysis.

In conclusion, the data show that long-term Th immune responses are heterogeneous in allo-BMT recipients as well as in their donors as shown by diverse TCR gene segment usage and amino acid composition of the CDR3. However, some individuals use TCR with similar TCR–CDR3 motifs, presumably reflecting recognition of similar unidentified epitopes of TT. Furthermore, we were able to show that, at least in part, these CD4+ Th cell responses were stable in time as was previously established for CD8+ cytotoxic T cell responses. These persistent TT-specific T cells that were present in the majority of the individuals may be important for the first line of defense upon renewed exposure to microbial antigens.


    Acknowledgments
 
We would like to thank Drs S. J. P Gobin and I. I. N. Doxiadis (Leiden University Medical Center, Leiden, The Netherlands) for critically reading the manuscript, and Charlotte Bigland and Rona M. Smith (Leiden University Medical Center) for technical assistance. This research was supported in part by the J. A. Cohen Institute for Radiopathology and Radiation Protection (IRS).


    Abbreviations
 
allo-BMT allogeneic bone marrow transplantation
APC antigen-presenting cells
BLCL B lymphoblastoid cell lines
BM bone marrow
CDR complementarity-determining region
PBMC peripheral blood mononuclear cell
SCID severe combined immunodeficiency
SI stimulation index
TT tetanus toxoid
TTX tetanus toxin

    Notes
 
Transmitting editor: J. Borst

Received 26 June 2000, accepted 5 January 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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