Haematologica 2002; 87:(10)ECR31
[Medline] [prev] [index] [next]Reconstitution of T-cell receptor repertoire diversity following non-myeloablative allogeneic stem cell transplantation in an acute myeloid leukemia patient.
Camillo Almici, Luisa Imberti*, Arnalda Lanfranchi,* Rosanna Verardi,* Marco Bellinzoni,* Silvia Berta,* Teodosio Izzi
Department of Internal Medicine-Hematology, BMT Unit, Spedali Civili di Brescia; *3° Servizio Analisi, Institute of Chemistry, University of Brescia, Italy.
Correspondence: Almici Camillo, MD Department of Internal Medicine-Hematology, BMT Unit, Spedali Civili di Brescia, Pz.le Spedali Civili,1. 25125 Brescia, Italy. Phone: +39.030.3996421. Fax +39.030.3995865. E-mail: almici@osp.unibs.it
Due to the poor engraftment of an autologous bone marrow transplant for acute myeloid leukemia, a 32-year old man progressively became red blood cell and platelet transfusion dependent. Repeated marrow examinations did not show any evidence of leukemia, but because of the hypo-aplasia and deteriorated condition he received, after non-myeloablative conditioning, his brother's unmanipulated G-CSF-primed peripheral blood stem cells. Besides confirming rapid and complete donor lineage specific chimerism, in order to give information about the dynamics of T lymphocyte engraftment after non-myeloablative stem cell transplant, we characterized T-cell receptor repertoire diversity. We found that the reconstitution of T-cell heterogeneity was complete but relatively slow and delayed as regard to the presence of complete donor chimerism.Allogeneic transplantation is a curative procedure in a number of hematologic malignancies, but its efficacy is mainly limited by conditioning regimen - related toxicity. Preliminary results using non-myeloablative conditioning regimens have confirmed that durable engraftment of allogeneic stem cells can be achieved with a marked reduction in the immediate post-transplantation toxicity.1-5 However, due to the low anti-tumor effect of the non-myeloablative regimens, there is a higher risk of graft rejection due to incomplete ablation of recipient immunity and an increased risk of disease progression. Therefore, in order to maintain a graft-versus-tumor effect, complete donor T-cell engraftment needs to be accomplished by modulating the patient's immunosuppressive therapy and by donor lymphocyte infusion.1 Recent animal studies have demonstrated that stem cell engraftment can occur using minimally myelotoxic conditioning regimens providing that post-transplant immunosuppressive therapy not only prevents graft-versus-host disease but also controls host-versus-graft reactions.6 While it is known that the reconstitution of a heterogeneous T-cell repertoire can be achieved after myeloablative therapy and allogeneic transplantation,7 the kinetics of T lymphocyte engraftment after non-myeloablative regimens has not yet been fully investigated. Therefore, we analyzed how the T-cell diversity was generated and maintained in an acute myeloid leukemia (AML) patient undergoing non-myeloablative stem cell transplant (NST) and in whom the post-grafting immunosuppression consisted of mycofenolate mofetil (MMF) and cyclosporine (CSP).
A patient. We describe a 32-year old man affected by AML (FAB M4) who, due to the progressive decrease in blood cell counts, at 10 months after autologous bone marrow transplantation (busulphan 16 mg/kg and cyclophosphamide 200 mg/Kg as conditioning regimen) became red blood cell and platelet transfusion dependent. Repeated marrow examinations showed hypo-aplasia without any evidence of leukemia and, therefore, he was referred for an unrelated allogeneic transplant that, because of recurrent bacterial and viral infections, was subsequently delayed. The deteriorated patient's condition made him ineligible for a conventional allogeneic procedure, and since his brother, HLA-A, -B, -DRB1 identical was considered as donor even though known to be anti-HCV positive, he underwent a NST.
Stem cell collection and transplant procedure. Peripheral blood stem cells (PBSC) mobilized by granulocyte colony-stimulating factor (G-CSF, 10 µg/Kg daily for 5 days) were collected from the donor by leukapheresis, and prepared for fresh infusion, without any further selection/depletion. The number of CD34+ and CD3+ cells in the leukapheresis product was measured by automated leukocyte counting and flowcytometry using FACScan (Becton Dickinson, Mountain View, CA, USA).
After a preparative regimen consisting of fludarabine alone (30 mg/ml, days -5 to -2; Fludara, Schering AG, Germany), the patient was reinfused with his brother's PBSC (4.6x106 CD34+ cells/Kg, 2.88x108 CD3+ cells/kg), followed by G-CSF (5 mg/Kg daily) therapy until engraftment; post-transplant immunosuppression consisted of MMF (15 mg/Kg twice daily, from day 0 to 30) and CSP (3 mg/kg/day intravenously, -1 to 20, and 6 mg/Kg twice a day orally until day +100).
Chimerism assay. Peripheral blood samples were collected before NST and at serial time points after transplant. Mononuclear cells were obtained by Ficoll-Hypaque gradient and CD3, CD19, CD14 positive subpopulations were prepared by magnetic beads separation. DNA preparation and chimerism assay were performed according to standard protocols. The tandem repeat polymorphism used was APOB, the 3' flanking region of the Apolipoprotein B gene, located in chromosome 2p24-p23.
Analysis of TCR repertoire. RNA and cDNA for the study of alpha/beta and gamma/delta T cell repertoires were prepared from CD4+ and CD4- subsets (purity > 95%) obtained by magnetic bead separation on day +126 and +262 post-NST. Primers, PCR conditions and procedures used for the analysis of the T-cell receptor (TCR) beta (TCRBV) and delta (TCRDV) chain usage have been elsewhere described.8 For heteroduplex analysis, PCR products, obtained after amplification with TCRBV- and TCRDV-specific primers, were heated to 95°C for 5 minutes, cooled to 50°C for 1 hour, and loaded on 12% non-denaturing polyacrylamide gel electrophoresis (PAGE, 29:1 acrylamide/bisacrylamide). Gels were run for 5-6 hours at 200 V at room temperature and stained, in the dark, for 30-60 minutes in a solution containing 0.75 mg/mL ethidium bromide in 200 mL of TBE.9 Homoduplex and heteroduplex bands (indicative of monoclonal and oligoclonal expansions, respectively) as well as the smears (resulting from the amplification of polyclonal populations) were detected by UVP's Gel Documentation System GDS8000 and analyzed using GelWorks 1D Analysis software. The appearance of dominant peaks indicates the presence of oligoclonal or clonal T-cell populations, while polyclonal T-cell populations are detected as more or less bent lines rising above the threshold.
Results. The engraftment was rapid, with absolute neutrophil count >500/mL on day 9 and platelets >20,000/mL and >50,000/mL on day 10 and 26, respectively (Figure 1), with no occurrence of acute graft-versus-host disease (GVHD). By analyzing VNTR APO B polymorphism, complete donor chimerism was detected by day +10 on granulocytes and by day +92 on lymphocytes, and subsequently confirmed on positively selected B-, T-cell and monocytic subpopulations (Figure 1). Now, at more than two years post-NST, the patient is in good condition, with stable engraftment, without any signs of chronic GVHD.
To determine whether donor T-cell engraftment was characterized by the presence of heterogeneous T-cell repertoires, we analyzed the diversity of T cells obtained from the donor, before PBSC collection, and from the patient at two different time points after transplantation. In these last two samples the pattern of TCRBV chain expression was similar (Figure 2A), but different from that of the donor (for instance, TCRBV14 segment was high in CD4- cells prepared from the recipient but low in CD4- lymphocytes from the donor). Since we previously found that the level of TCRBV heterogeneity is not necessarily related to the relative percentage of TCRBV expression,10 we also performed a heteroduplex analysis of the most represented TCRBV chains (Figure 2B). With this method amplified material from polyclonal lymphoid cells migrates on the polyacrylamide gel as a smear, composed of different sized PCR fragments, while the mismatched chains, derived from oligoclonal populations, migrate as more or less fairly discrete heteroduplex bands of different size, which can be separated from the matched homoduplex bands obtained from homogeneous clonal cells. When analyzed with appropriate software, homoduplex and heteroduplex bands result in peaks of different sizes, indicating the presence of clonal or oligoclonal T-cell populations. The absence of relevant peaks suggests the absence of cells using a specific subfamily of TCR genes. The data showed that in the donor CD4+ and CD4- lymphocytes give rise to few small peaks, indicating minor oligoclonal expansions, which can be the result of chronic stimulation by HCV infection (fig. 2B). In contrast, the presence of several discrete peaks in lymphocyte subsets prepared on day +126 from the patients is indicative of a highly restricted T cell diversity. Most of these peaks were not detected on day +262, suggesting an enlargement of T cell heterogeneity in both T cell subsets. The pattern of TCR gamma/delta lymphocyte diversity was identical in all samples analyzed (Figure 2C).
Discussion. The diversity of the TCR repertoire is an essential component of immunocompetence and provides an indicator of immune reconstitution post-transplant. It has been reported that after conventional allogeneic transplant, with myeloablative conditioning, T-cell reconstitution is initially derived from the expansion of mature donor T-cells and restoration of T-cell repertoire diversity occurs, starting at 6 months after transplant, only in patients who have achieved complete donor hematopoiesis;7 however, how the T-cell repertoire is generated and maintained after NST has been little investigated. Therefore, in order to gain information about the characteristics of the T cell engraftment after NST, we analyzed the diversity of the TCR repertoire in samples obtained 4 and 9 months post-NST. In the first sample, even in the presence of complete donor chimerism, the patient showed a marked reduction of TCR diversity, with most TCRBV families demonstrating monoclonal or oligoclonal profiles, consistent with the expansion of distinct T-cell populations. Since the patient had no evidence of ongoing infection (he remained HCV-) and there were no major pre-existing clonal expansions in the donor, the reason for the limited TCR repertoire diversity could be that transfused lymphocytes with particular TCRBV subfamilies may preferentially proliferate over the others in the host, probably as the result of alloantigenic stimulation. A normalization of repertoire was observed in the sample obtained 9 months after NST, in which the profiles of both CD4+ and CD4- were comparable to that of the donor. It is of interest that the TCRDV repertoire was identical in the two samples obtained at the two different time points from the recipient and that the TCRBV diversity profiles are superimposable in both donor and recipient. These data suggest that in our patient, as already demonstrated in patients after myeloablative conditioning,7 the reconstitution of T-cell diversity was complete but relatively slow, and was delayed until a complete donor hematopoiesis had been obtained.
Furthermore these results indicate that the generation and maintenance of diversified alpha/beta and gamma/delta T cells may follow different paths, or they are possible only in the presence of heterogeneous repertoires in the donor.
In conclusion, in our patient a non-myeloablative strategy allowed rapid engraftment of donor cells, prompt hematologic recovery and complete reconstitution of normal T-cell immunity. Therefore, NST could represent a promising alternative strategy in patients considered ineligible for conventional allogeneic transplant because of advanced age or co-morbidities, allowing the reconstitution of T-cell repertoire diversity was well as guaranteeing myeloid engraftment.References
1) Champlin R, Khouri I, Shimoni A,Gajewski J, Kornblau S, Molldrem J, et al. Harnessing graft-versus-malignancy: non-myeloablative preparative regimens for allogeneic haematopoietic transplantation, an evolving strategy for adoptive immunotherapy. Br J Haematol 2000; 111: 18-29.
2) Childs R, Clave E, Contentin N, Jayasekera D, Hensel N, Leitman S, et al. Engraftment kinetics after nonmyeloablative allogeneic peripheral blood stem cell transplantation: full donor T-cell chimerism precedes alloimmune responses. Blood 1999; 94: 3234-41.
3) Giralt S, Estey E, Albitar M, van Besien K, Rondon G, Anderlini P, et al. Engraftment of allogeneic hematopoietic progenitor cells with purine analog-containing chemotherapy: harnessing graft-versus-leukemia without myeloablative therapy. Blood 1997; 89: 4531-6.
4) Slavin S, Nagler A, Naparstek E, Kapelushnik Y, Aker M, Cividalli G, et al. Nonmyeloablative stem cell transplantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and nonmalignant hematologic diseases. Blood 1998; 91: 756-63.
5) Khouri IF, Keating M, Korbling M, Przepiorka D, Anderlini P, O'Brien S, et al. Transplant-Lite: induction of graft-versus-malignancy using fludarabine-based nonablative chemotherapy and allogeneic blood progenitor-cell transplantation as treatment for lymphoid malignancies. J Clin Oncol 1998; 16: 2817-24.
6) Storb R, Cong Y, Zaucha JM, Deeg J, Georges G, Kiem HP, et al. Stable mixed hematopoietic chimerism in dogs given donor antigen, CTLA4Ig, and 100cGy total body irradiation before and pharmacologic immunosuppression after marrow transplant. Blood 1999; 94: 2523-9.
7) Wu CJ, Chillemi A, Alyea EP, Orsini E, Neuberg D, Soiffer RJ, et al. Reconstitution of T-cell receptor repertoire diversity following T-cell depleted allogeneic bone marrow transplantation is related to hematopoietic chimerism. Blood 2000; 95: 352-9.
8) Signorini S, Imberti L, Pirovano S et al. Intrathymic restriction and peripheral expansions of T-cell repertoire in Omenn Syndrome. Blood 1999; 10: 1-13.
9) Sottini A, Quiros-Roldan E, Albertini A et al. Assessment of T-cell receptor beta chain diversity by heteroduplex analysis. Human Immunol 1996; 48: 12-22.
10) Villa A, Santagata S, Bozzi F et al. Partial V(D)J recombination activity leads to Omenn syndrome. Cell 1998; 93: 886-96.