We have previously reported that skewed repertoires of T cell receptor-β chain variable region (TCRBV) and TCR-α chain variable region (TCRAV) are observed at an early period after allogeneic hematopoietic cell transplantation. Furthermore, we found that T lymphocytes using TCRBV24S1 were increased in 28% of the recipients of allogeneic grafts and an increase of TCRBV24S1 usage was shown to result from clonal expansions. Interestingly, the arginine residue was frequently present at the 3′ terminal of BV24S1 segment and was followed by an acidic amino acid residue within the CDR3 region. These results suggest that these clonally expanded T cells are not randomly selected, but are expanded by stimulation with specific antigens. This study was undertaken to elucidate the mechanisms of the post-transplant skewing of TCR repertoires. Since the CD8+CD28−CD57+ T cell subset has been reported to expand in the peripheral blood of patients receiving allogeneic hematopoietic cell grafts, we examined the TCRAV and TCRBV repertoires of the CD8+CD28− T cell and CD8+CD28+ T cell subsets, and also determined the clonality of both T cell populations. In all three recipients examined, the CD8+CD28− T cell subset appeared to define the post-transplant TCR repertoire of circulating blood T cells. Moreover, the CDR3 length of TCRBV imposed constraints in both CD8+CD28− T cell and CD8+CD28+ T cell subsets. The DNA sequences of the CDR3 region were determined, and the same clones were identified within both CD8+CD28− and CD8+CD28+ T cell subsets in the same individuals. These results suggest that the clonally expanded CD8+CD28− T cells after allogeneic hematopoietic cell transplantation derive from the CD8+CD28+ T cell subset, possibly by an antigen-driven mechanism, resulting in the skewed TCR repertoire. Bone Marrow Transplantation (2001) 27, 731–739.
The CD28 molecule is a disulfide-linked homodimer expressed on the surface of T cells and binds to the natural ligand B7 family members, CD80/CD86, expressed on antigen-presenting cells, resulting in costimulation of T cell activation.1 In humans, all thymocytes and the vast majority of peripheral blood T cells at birth express CD28. The proportion of CD8+ T cells lacking CD28 expression increases during human aging, so that approximately 30% of CD8+ T cells are negative for CD28 in adults.2 Most of the CD8+CD28− T cells express CD57.2 A number of reports have shown that the proportion of CD8+CD28− T cells is increased in HIV-infected patients.34
The CD8+CD28−CD57+ T cell subset is also expanded in the peripheral blood of patients receiving allogeneic bone marrow transplants.56 However, the mechanisms and biological relevance of this expansion remain to be elucidated. Moreover, it is still uncertain whether the CD8+CD28− T cell subset arises from the CD8+CD28+ T lymphocytes or whether both subsets belong to distinct T cell lineages.
We have previously reported that the skewed TCRAV and TCRBV repertoires are observed at an early period after allogeneic hematopoietic cell transplantation.7 We also found that the TCR usage appeared to be limited and T cells using TCRBV24S1 were increased in 28% of the patients receiving allogeneic hematopoietic cell grafts. An increase of the TCRBV24S1 usage was shown to be the result of clonal T cell expansions. Interestingly, in four out of six patients examined, the arginine residue was present at the 3′ terminal of BV24S1 segment and was followed by an acidic amino acid residue, such as glutamic acid and aspartic acid, within the CDR3 regions. These results suggest that these clonally expanded T cells are not randomly selected, but are expanded by stimulation with specific antigens. Further evidence in support of this includes oligoclonal expansion of CD8+CD57+ T lymphocytes previously reported in marrow transplant recipients.89 If the clonally expanded CD8+CD28− T cells following transplantation originated from CD8+CD28+ T cells that have been exposed to certain antigens, it is possible that identical T cell clones could be demonstrated in both CD28− and CD28+ subsets. In this study, we present evidence for this hypothesis and discuss the biological relevance of our findings.
Materials and methods
Informed consent was obtained from the patients and donors before blood samples were collected. All the patients were conditioned with myeloablative chemoradiotherapy, mostly consisting of fractionated total body irradiation (12 Gy in six fractions) and cyclophosphamide (60 mg/kg/day for 2 days), followed by infusion of allogeneic marrow or blood stem cell grafts from HLA-matched donors. All the patients received cyclosporin A and short-term methotrexate for prophylaxis of acute graft-versus-host disease (GVHD).10 Engraftment was achieved in patients, and confirmed by recovery of hematopoiesis and the presence of donor-derived sex chromosome or mismatched antigens on red cells. Clinical grading of acute GVHD was determined according to the criteria reported by Glucksberg et al.11 Patients were monitored for cytomegalovirus (CMV) infection by weekly CMV antigenemia assays12 from when the granulocyte count reached 500/μl until day 100 after transplantation. Patients who were positive for CMV antigenemia received prophylactic ganciclovir (5 mg/kg/day, 3 days a week) from when the granulocyte count was greater than 1000/μl.13
Peripheral blood mononuclear cells (PBMCs) were isolated by the Ficoll/Conray gradient centrifugation method from heparinized blood. PBMCs were stained with FITC- or PE-conjugated monoclonal antibodies and analyzed using a flow cytometer (Cytron Absolute, Ortho Diagnostics, Tokyo, Japan). Monoclonal antibodies used in this study were as follows: anti-CD3 (SK7, IgG1; Becton Dickinson, San Jose, CA, USA); anti-CD4 (MT310, IgG1; DAKO, Glostrup, Denmark); anti-CD8 (DK25, IgG1; DAKO); anti-CD28 (CD28.2, IgG1; Beckman Coulter, Fullerton, CA, USA); anti-CD57 (HNK-1, IgM; Beckman Coulter); anti-HLA-DR (L243, IgG2a; Becton Dickinson); anti-CD25 (ACT-1, IgG1; DAKO); anti-CD122 (TU27, IgG1; Becton Dickinson); and control mouse IgG (X40, IgG1; DAKO). In some experiments, CD4+CD28+, CD4+CD28−, CD8+CD28+ and CD8+CD28− T cell subsets were collected by FACS sorting.14 Each population was >98% pure.
TCRAV and TCRBV repertoire analysis
Analysis of TCRAV and TCRBV repertoires was performed by an adaptor ligation PCR-based microplate hybridization assay, as reported previously.15 Briefly, total RNA was extracted from PBMCs and converted to double-strand cDNA using the SuperScript cDNA synthesis kit (BRL, Bethesda, MD, USA). The P10EA/P20EA adaptors were ligated to the 5′ end of cDNA prepared from PBMCs, and PCR was performed using either a biotinylated TCRCA-specific or TCRCB-specific primer, and a P20EA primer. Biotinylated PCR products were hybridized with immobilized TCRAV- or TCRBV-specific primers in 96-well microtiter plates. Subsequently, alkaline phosphatase-conjugated streptavidin was added to each well and a colorimetric assay was performed. Skewing of the TCR repertoire was defined as a significant increase of TCRAV and TCRBV subfamilies with greater percentage than the mean plus 3 standard deviation (s.d.) of 20 healthy individuals, and exceeded 5% of total circulating blood T cells.715
PCR amplification and CDR3 size distribution analysis of the TCR-β chain
The procedure for CDR3 size analysis (spectratyping) for the TCR-β chain has been described elsewhere.161718 Total RNA was extracted from PBMCs using a RNeasy Total RNA Kit (Qiagen, Hilden, Germany) and was used for first-strand cDNA synthesis with an oligo-dT primer (First-Strand cDNA Synthesis Kit, Amersham Pharmacia Biotech, Uppsala, Sweden).1920 Aliquots of the cDNA were amplified with a Vβ-specific primer and a Cβ-specific primer. Primer sequences were previously described.1821 PCR amplification was performed for 40 cycles in a 20 μl reaction mixture containing 0.2 μM of each primer and 0.5 U of Taq polymerase (TaKaRa, Osaka, Japan). Conditions for the PCR were as follows: denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1.5 min. Following the 40 cycles of PCR, an additional extension at 72°C for 15 min was performed. The PCR buffer was 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, and 0.2 mM of each dNTP.
Aliquots (4 μl) of the unlabeled V β–Cβ PCR products were subjected to one cycle of elongation (runoff reaction) with a FAM-labeled nested Cβ primer (FAM-CB3) under the following conditions: denaturation at 94°C for 2 min, annealing at 55°C for 1 min, and extension at 72°C for 15 min. The reaction buffer was the same as that described above. The labeled PCR products were mixed with the size marker (GeneScan-500 TAMRA; Applied Biosystems, Warrington, UK), and loaded on to 5% polyacrylamide sequencing gels for determination of size and fluorescence intensity using an automated DNA sequencer (ABI 377, Perkin-Elmer Applied Biosystems, Foster City, CA, USA). Data were analyzed using GeneScan software (Perkin-Elmer Applied Biosystems).
Sequencing of CDR3 region in the TCR-β chain
PCR products of the TCR-β chain were cloned into the PCR2.1 TA cloning vector (Invitrogen, Carlsbad, CA, USA) and sequenced using a Big-Dye Terminator Cycle Sequencing Kit (Perkin-Elmer Applied Biosystems).22 Sequence analysis was performed using an Applied Biosystems 377A automatic DNA sequencer.
CD8+CD28− T cells as an initially repopulated T cell subset after allogeneic bone marrow transplantation
During the first few months after transplant the recipients show lymphocytopenia and reduced CD4 counts. In agreement with previous reports, there was a marked increase of CD28− T cell fraction early after bone marrow transplantation (Figure 1c). Since the CD4/CD8 ratio is invariably low at this time, the majority of the T cells during the early immune reconstitution have the CD8+CD28− phenotype. We have previously reported that the skewed TCRAV and TCRBV repertoires are observed at an early period after allogeneic hematopoietic cell transplantation.7 We therefore asked whether the TCR repertoire of CD8+CD28− T cell subset would impose a skew upon the post-transplant TCR repertoire of circulating blood T cells.
TCRAV and TCRBV repertoires of the CD8+CD28− T cell subset defines the skewed TCR repertoire of circulating T lymphocytes
PBMCs were collected from three recipients of allogeneic hematopoietic cell grafts, and the T cells were separated into four subsets by FACS sorting depending upon their expression of CD4, CD8 and CD28. TCRAV and TCRBV repertoires were analyzed on PBMC and these four T cell subsets. Consistent with our previous report,7 all three patients showed skewed TCRAV and TCRBV repertoires at an early phase of engraftment. Results of one representative patient are shown in Figure 2. In this patient, the TCR VA3–1, VA14–1, VB15–1, VB22–1 and VB24–1 subfamilies were increased following transplantation, although there was no difference in TCR repertoires between the donor and the recipient pre-transplant. The CD8+CD28− T cell subset was the major population of the T cells in this patient (Table 1). The TCRAV and TCRBV repertoires of the CD8+CD28− T cell subset were most similar to those of the PBMCs among other subsets, and the increased TCRAV and BV subfamilies were enriched in this population (Figure 3). In the same manner, two other patients were examined and similar results were found (Table 1, Figure 4). Moreover, a skew of the TCR repertoire in the CD8+CD28− T cell subset was found within both TCRAV and TCRBV. Again, these results suggest that the expansion of CD8+CD28− T cells was the result of recognizing antigens in the context of MHC/peptide but not the way of recognition of superantigens.23
Clonal diversity of CD28+ and CD28− T cell subsets with skewed TCRBV repertoire
If the expansion of CD8+CD28− T lymphocytes is imposed by the stimulation with classical peptide antigens, one would expect to find CDR3 length constraints.24 We measured the CDR3 size of skewed TCRBV in each T cell subset. Clearly, the expansion of CD8+CD28− T cells was clonal or oligoclonal (Figure 5), which supports the hypothesis mentioned above that the post-transplant expansion of CD8+CD28− T cells was antigen-driven.2425 In addition, the diversity of the CD8+CD28+ T cell subset was also restricted as well as the CD8+CD28− T cell population (Figure 5). The CDR3 length of the dominant peak was similar in both CD8+CD28+ and CD8+CD28− T cells in a given patient, suggesting that there may exist identical clones within the two subsets.
Demonstration of identical clones within both CD8+CD28− and CD8+CD28+ T cell subsets
If the CD8+CD28− T cells originated from the CD8+CD28+ T cell subset, one would expect the identification of the same clones within both subsets. We subcloned PCR products of skewed TCR-β chains to see if the identical CDR3 sequences could be found in both CD8+CD28− and CD8+CD28+ subsets. We found identical clones in both subsets in all three patients examined (Table 2).
The present study demonstrates the presence of identical expanded clones within both CD8+D28+ and CD8+CD28− T cell subsets in recipients of allogeneic hematopoietic stem cell grafts. Our observation suggests that the clonally expanded CD8+CD28− T cells following allogeneic blood and marrow transplantation originate from the CD8+CD28+ T cells that have been exposed to antigens. The transition between the two subsets could be unidirectional, since CD8+CD28− T lymphocytes are infrequent in cord blood, thymus and lymph nodes but this subset increases during human aging.27 CD8+CD28− T cells have been demonstrated to have shorter telomeres than CD8+CD28+ T cells.28 Moreover, it has been recently reported that CD8+CD28− T cells can be generated from chronically stimulated CD8+CD28+ T cells.29 Mugnaini et al30 have also reported the presence of identical expanded clones within both CD8+CD28− and CD8+CD28+ T cell fractions in HIV patients. Thus, it has become more evident that CD8+CD28− and CD8+CD28+ T cell subsets belong to the same T cell lineage with just a distinct phenotype.
We have previously observed the skewed TCRAV and TCRBV repertoires at an early period after human allogeneic blood and marrow transplantation.7 We have also found that there was a strong correlation between delta scores of TCRAV and those of TCRBV. Delta scores signify the extent of alteration of the TCR repertoire.31 In 28% recipients of allogeneic grafts, there was a clonal expansion of the T cells carrying TCRBV24S1. DNA sequence analysis revealed that an arginine residue followed by acidic amino acid residues is frequently observed in the CDR3 of TCR-β chain containing BV24S1, suggesting that skewing of the TCR repertoire is antigen-driven. To support this, measurement of the CDR3 length of TCRBV clearly demonstrated the clonal expansions in the CD8+CD28− T cell subset in our study. Since the CD8+CD28− T cell fraction is the major population among T cells during the first several months after transplantation, the TCR repertoire of this subset imposes a great impact on the repertoire of circulating blood T lymphocytes.
Antigen specificity of the clonally expanded CD8+CD28− T cells following allogeneic transplantation remains to be determined. The CD8+CD28− T cell population has been shown to contain virus-specific memory cytotoxic T lymphocytes that respond to human cytomegalovirus (CMV) and human immunodeficiency virus (HIV).323334353637 Thus, clonally expanded CD8+CD28− T cells following transplantation may be derived from the T cells recognizing antigens that persistently exist in the host, and may contain CTL clones specific for viral antigens such as herpesviruses.
Post-transplant immune reconstitution should be also addressed at an antigen-specific level. Quantitative analysis of antigen-specific T cells has now become feasible by using fluorescence-conjugated major histocompatibility complex (MHC)-peptide tetramers, although this approach is limited to patients with certain HLA types.3839 Antigen specificity of the clonally expanded CD8+CD28− T cells following allogeneic transplantation may be determined by taking advantage of this technique.
As yet, we are unable to draw any conclusions regarding the relationship between the magnitude of clonal expansion of CD8+CD28− T cells and the immune status of blood and marrow transplant recipients. Quantitative analysis of antigen-specific CD8+ T cells may provide insights into our understanding of clonal expansions of T cells following allogeneic hematopoietic cell transplantation.
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We are grateful to the hematology staff at Akita University Medical Center for their treatment of the patients included in this study. This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan (Grant No. 08670508, 10670932), the Yamashita Taro-Kensho Memorial Foundation and the Uehara Memorial Foundation to MH.
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Horiuchi, T., Hirokawa, M., Kawabata, Y. et al. Identification of the T cell clones expanding within both CD8+CD28+ and CD8+CD28− T cell subsets in recipients of allogeneic hematopoietic cell grafts and its implication in post-transplant skewing of T cell receptor repertoire. Bone Marrow Transplant 27, 731–739 (2001) doi:10.1038/sj.bmt.1702859
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