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Prepublished online as a Blood First Edition Paper on May 13, 2002; DOI 10.1182/blood-2001-11-0005. This Article Abstract Full Text (PDF) All Versions of this Article: 2001-11-0005v1 100/5/1915    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Eyrich, M. Articles by Schlegel, P. G. Search for Related Content PubMed PubMed Citation Articles by Eyrich, M. Articles by Schlegel, P. G. Related Collections Immunobiology Brief Reports Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 September 2002, Vol. 100, No. 5, pp. 1915-1918 BRIEF REPORT Distinct contributions of CD4+ and CD8+ naive and memory T-cell subsets to overall T-cell-receptor repertoire complexity following transplantation of T-cell-depleted CD34-selected hematopoietic progenitor cells from unrelated donors Matthias Eyrich, Tanja Croner, Christine Leiler, Peter Lang, Peter Bader, Thomas Klingebiel, Dietrich Niethammer, and Paul G. Schlegel From the Pediatric Stem Cell Transplant Program, University of Tuebingen, and the University Children's Hospital, Department of Pediatric Hematology/Oncology, University of Frankfurt, Germany.     Abstract Top Abstract Introduction Study design Results and discussion References Normalization of restricted T-cell-receptor (TCR) repertoire is critical following T-cell-depleted (TCD) stem cell transplantation. We present a prospective study analyzing respective contributions of naive and memory T-cell subsets within the CD4+ and CD8+ compartments to the evolution of overall TCR-repertoire complexity following transplantation of CD34-selected peripheral blood progenitor cells from unrelated donors. During the first year after transplantation, sorted CD4/45RA, CD4/45R0, CD8/45RA, and CD8/45R0 subsets were analyzed at 3-month intervals for TCR-repertoire complexity by CDR3 size spectratyping. Skew in TCR-repertoire was observed only in early memory-type T cells. CD4+ and CD8+ subsets differed in clonal distribution of CDR3 sizes, with rapid Gaussian normalization of bands in CD4/45R0+ T cells. Naive T cells displayed normal repertoire complexity and contributed significantly to skew correction. Our data provide direct evidence for an important role of de novo maturation of naive T cells in normalization of an initially restricted TCR-repertoire following transplantation of CD34-selected, TCD-depleted peripheral blood progenitors from unrelated donors. (Blood. 2002;100:1915-1918) © 2002 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References Reconstitution of a functional T-cell compartment after allogeneic stem cell transplantation (allo-SCT) requires not only normalization of peripheral T-cell numbers but also restoration of a diverse T-cell-receptor (TCR) repertoire.1 Several studies in recent years have demonstrated only minor abnormalities in TCR repertoire diversity following transplantation of unmanipulated bone marrow from HLA-identical related donors,2,3 whereas transplantation from unrelated or HLA-mismatched related donors, particularly after T-cell depletion (TCD), results in severe skewing of the TCR repertoire during the first posttransplantation year.2-5 It has been suggested that in these transplantations, reconstitution of a normal TCR repertoire relies on de novo generation of naive T cells and requires residual thymic function of the host.1,6,7 However, the relative contribution of naive and memory T-cell subsets to overall TCR repertoire diversity after allo-SCT has not been investigated thus far. In this study, we prospectively analyzed TCR repertoire diversity of CD4+ and CD8+ naive and memory T cells at 3-month intervals in 10 children during the first year after allo-SCT from unrelated donors. The results demonstrate a skewing of the repertoire during the first 6 months after transplantation limited to the memory pool of CD4+ and CD8+ T-cell subsets. In contrast, naive CD4+ and CD8+ T cells exhibit an ongoing diverse TCR repertoire from their earliest detection. Memory-type CD4+ cells tend to normalize their patterns of clonal distribution more rapidly than memory-type CD8+ cells, in which a pattern of clonal dominance can be observed beyond the first year after transplantation. Our results provide direct evidence of corrections in gaps and skewing in the initial post-allo-SCT-restricted TCR repertoire by naive CD4+ and CD8+ T cells and emphasize an important role of de novo maturation of T cells through the thymus for complete reconstitution of the T-cell compartment.     Study design Top Abstract Introduction Study design Results and discussion References Ten children underwent transplantation with TCD CD34+-selected peripheral blood stem cells from unrelated donors (Table 1). The study was approved by the University of Tuebingen's institutional review board, and informed consent was obtained from the parents. Graft-versus-host disease (GVHD) prophylaxis was achieved by TCD of the graft using the CliniMACS system (Miltenyi, Bergisch-Gladbach, Germany) without posttransplantation immunosuppression. Engraftment as determined by absolute neutrophil count (ANC) greater than 500/µL over 3 consecutive days was achieved after a median of 13.5 days (range, 9-24 days).                                View this table: [in this window] [in a new window]   Table 1. Patient characteristics Peripheral blood mononuclear cells (PBMCs) were collected at 3-month intervals using standard procedures. In the early posttransplantation phase, when memory T cells dominated the peripheral T-cell pool, PBMCs were MACS-sorted into CD4+ and CD8+ cells following the manufacturer's protocol, and purity was confirmed by FACS analysis. When more than 10% of the CD4+/CD45RA+ naive-type T cells were detectable in the peripheral blood, PBMCs were sorted into naive (CD45RA+) and memory (CD45RO+) CD4+ and CD8+ cells using the CD4+ Multisort Kit (Miltenyi). TCR repertoire diversity in the subsets was assessed by CDR3 size spectratyping of V1-24, as described previously.8 All cDNAs were controlled using a C-specific primer pair to ensure appropriate quality and quantity before they were subjected to spectratype analysis. Because a normal spectratype has been shown to consist of 5 to 8 bands per family with a Gaussian size distribution of TCR fragments9,10 scoring and size distribution were used to assess the normalization of TCR repertoire. In addition, TCR-repertoire complexity of T-cell subsets was determined in healthy controls (n = 4).     Results and discussion Top Abstract Introduction Study design Results and discussion References Previous longitudinal CDR3 size spectratype analyses in healthy persons have confirmed the TCR repertoire to be stable over time with a Gaussian distribution of TCR fragments and a median of 6 to 8 bands in each V-family.9,11 Furthermore, complexity and clonal distribution were reported to be equivalent in CD4+ and CD8+ T cells in healthy persons, with minor skews in certain CD8+ V families.12 However, in the early posttransplantation phase, before day 200, characterized by an expansion of only mature memory-type T cells, we observed a markedly restricted TCR repertoire, with different clonotypes identified in CD4+ and CD8+ T cells (Figure 1A). Restricted overall TCR diversity has also been reported by other investigators after the transplantation of autologous or allogeneic TCD grafts3,4,10 and can be explained by the low number of cotransplanted T cells in TCD grafts. The identification of different clonotypes in CD4+ and CD8+ subsets demonstrates that these populations are efficiently separated using the MACS technique (median purity, 80.6%). It should be noted that the skew observed in our patients was not caused by limited numbers of T cells available for analysis. In titration experiments we had previously determined the lower threshold of our assay to be in the range of 104 T cells.5 All samples in this study contained T-cell numbers above this limit (median, 6.8 × 105 CD3+ T cells; range, 0.1-149 × 105 CD3+ T cells). View larger version (53K): [in this window] [in a new window]   Figure 1. Distinct CDR3 size distributions in CD4+ and CD8+ naive and memory T-cell subsets. (A) Different CDR3 size fragments identified in CD4+ (top lane) and CD8+ (bottom lane) T-cell subsets. Representative example of V3, V5.1, and V13 families in the sorted subfractions of a patient with acute lymphoid leukemia (UPN 442) on day 147 after transplantation. (B) Increase in the median number of bands per V family in naive and memory CD4+ and CD8+ T-cell subsets after transplantation. Before day 200, no naive-type T cells were available for analysis. In parallel with the appearance of naive-type T cells in the periphery, the mean number of bands per V family of the CD4+ and CD8+ memory compartment significantly increased between the study time points, days 101 to 200 and days 201 to 300 (P < .001, Mann-Whitney U test). In the first 200 days after transplantation, peripheral T cells almost exclusively expressed a memory phenotype (CD45RO+ for CD4+, CD27+ for CD8+) (data not shown). It is generally assumed that the antigenic milieu of the host stimulates expansion of selected donor-derived memory T-cell clones that were cotransplanted with the graft,13 though several groups reported the persistence of recipient-derived clones in these subsets.1,10 Two to 5 patients in our cohort showed mixed donor-recipient chimerism during follow-up, with a median percentage of 2.5% (Table 2). Therefore, it seems possible that in these patients, expanded recipient clones might have contributed in part to TCR repertoire diversity.                                View this table: [in this window] [in a new window]   Table 2. Chimerism and TCR V repertoire reconstitution Between day 100 and day 200, repertoire complexity of the memory T-cell pool only marginally improved (Figure 1B; Table 2), suggesting that TCR repertoire complexity after TCD transplantation does not normalize without the addition of new clones either by adoptive transfer or by de novo maturation. However, we were able to detect differences between the CD4+ and CD8+ T-cell compartments. CD4+ memory-type T cells showed a faster normalization toward a Gaussian distribution of TCR fragments within the V families (Figure 1A), even before detection of naive T cells in the peripheral blood. This suggests that homeostatic proliferation14,15 of early naive T cells with subsequent acquisition of a memory phenotype might be operative in this setting. The generation of functional CD4+ memory T cells after encounter with endogenous antigen only has been described recently in TCR-transgenic mice.14 Although this phenomenon has also been reported in CD8+ naive T cells,15 we found it to be more relevant in the CD4+ compartment. Clonal dominance of certain V families in memory CD8+ T cells of transplant recipients persisted well beyond the first posttransplantation year and could be observed in healthy controls as well (data not shown). This pattern has been described in both populations12,16,17 and seems to be a characteristic feature of this T-cell subset. Newly appearing naive-type T cells emerging after day 200 showed a TCR repertoire comparable to that of healthy controls with a Gaussian distribution of TCR fragments (Figure 1B; Table 2). In parallel with the appearance of naive-type T cells in the periphery, repertoire complexity of the CD4+ and CD8+ memory pool significantly improved (P < .001, Mann-Whitney U test; Figure 1B), probably because of the transition of new naive T cells into a memory phenotype. None of the 10 children underwent donor lymphocyte infusion (DLI) during the study period; therefore, the transfer of mature T-cell clones by DLI can be excluded. Because the generation of naive T cells after allo-SCT seems to require residual thymic function, at least in CD4+ T cells,18,19 this underscores the importance of thymic involvement for the reconstitution of a complex TCR repertoire after TCD stem cell transplantation. The results of our study demonstrate that restricted TCR repertoire diversity after allo-SCT of TCD grafts from unrelated donors is limited to the early-appearing memory T-cell pool. Naive-type T cells, which appear several months after transplantation, display a normal TCR repertoire complexity and help to correct the initial skewing. Additional studies will be aimed at enhancing the generation of naive-type T cells, either by the administration of specific cytokines such as interleukin-720,21 or by protection of the thymus from chemotherapy- and radiotherapy-induced toxicity.     Acknowledgments We thank the staff of the BMT unit and outpatient department and the technical staff of the stem cell processing unit at the University Children's Hospital, Tuebingen for their excellent and dedicated patient care. We also thank Shangara Lal for critically reviewing the manuscript.     Footnotes Submitted December 18, 2001; accepted April 15, 2002. Prepublished online as Blood First Edition Paper, May 13, 2002; DOI 10.1182/blood-2001-11-0005. Supported by a grant from the fortuene-Program of the University of Tuebingen (no. 587/1999) and in part by the Deutsche Forschungsgemeinschaft Program Project Grant SFB 510-C4 (P.G.S. and D.N.) M.E. is a postdoctoral fellow of the Else Uebelmesser Foundation for Cancer Research (1.3-0415.221.18-03/97). The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Paul G. Schlegel, Pediatric Stem Cell Transplant Program, Program Project Grant IZKF, Hoppe-Seyler-Strasse 1, Tuebingen, D-72076, Germany; e-mail: p-g.schlegel{at}med.uni-tuebingen.de.     References Top Abstract Introduction Study design Results and discussion References 1. Roux E, Dumont-Girard F, Starobinski M, et al. Recovery of immune reactivity after T-cell-depleted bone marrow transplantation depends on thymic activity. Blood. 2000;96:2299-2303[Abstract/Free Full Text]. 2. Roux E, Helg C, Chapuis B, et al. T-cell repertoire complexity after allogeneic bone marrow transplantation. Hum Immunol. 1996;48:135-138[CrossRef][Medline] [Order article via Infotrieve]. 3. Godthelp BC, van Tol MJ, Vossen JM, et al. T-cell immune reconstitution in pediatric leukemia patients after allogeneic bone marrow transplantation with T-cell-depleted or unmanipulated grafts: evaluation of overall and antigen-specific T-cell repertoires. Blood. 1999;94:4358-4369[Abstract/Free Full Text]. 4. Bomberger C, Singh-Jairam M, Rodey G, et al. Lymphoid reconstitution after autologous PBSC transplantation with FACS-sorted CD34+ hematopoietic progenitors. Blood. 1998;91:2588-2600[Abstract/Free Full Text]. 5. Eyrich M, Lang P, Lal S, et al. A prospective analysis of the pattern of immune reconstitution in a paediatric cohort following transplantation of positively selected human leucocyte antigen-disparate haematopoietic stem cells from parental donors. Br J Haematol. 2001;114:422-432[CrossRef][Medline] [Order article via Infotrieve]. 6. Mackall CL, Gress RE. Pathways of T-cell regeneration in mice and humans: implications for bone marrow transplantation and immunotherapy. Immunol Rev. 1997;157:61-72[CrossRef][Medline] [Order article via Infotrieve]. 7. Douek DC, Vescio RA, Betts MR, et al. Assessment of thymic output in adults after haematopoietic stem-cell transplantation and prediction of T-cell reconstitution. Lancet. 2000;355:1875-1881[CrossRef][Medline] [Order article via Infotrieve]. 8. Gorski J, Piatek T, Yassai M, et al. Improvements in repertoire analysis by CDR3 size spectratyping: bifamily PCR. Ann N Y Acad Sci. 1995;756:99-102[Medline] [Order article via Infotrieve]. 9. Gorski J, Yassai M, Zhu X, et al. Circulating T cell repertoire complexity in normal individuals and bone marrow recipients analyzed by CDR3 size spectratyping: correlation with immune status. J Immunol. 1994;152:5109-5119[Abstract]. 10. Wu C, Chillemi A, Alyea E, et al. Reconstitution of T-cell receptor repertoire diversity following T-cell depleted allogeneic bone marrow transplantation is related to hematopoietic chimerism. Blood. 2000;95:352-359[Abstract/Free Full Text]. 11. Tanchot C, Rocha B. The organization of mature T-cell pools. Immunol Today. 1998;19:575-579[CrossRef][Medline] [Order article via Infotrieve]. 12. Naumov YN, Naumova EN, Gorski J. CD4+ and CD8+ circulating alpha/beta T-cell repertoires are equally complex and are characterized by different levels of steady-state TCR expression. Hum Immunol. 1996;48:52-62[CrossRef][Medline] [Order article via Infotrieve]. 13. Mackall CL, Bare CV, Granger LA, et al. Thymic-independent T cell regeneration occurs via antigen-driven expansion of peripheral T cells resulting in a repertoire that is limited in diversity and prone to skewing. J Immunol. 1996;156:4609-4616[Abstract]. 14. Gudmundsdottir H, Turka LA. A closer look at homeostatic proliferation of CD4+ T cells: costimulatory requirements and role in memory formation. J Immunol. 2001;167:3699-3707[Abstract/Free Full Text]. 15. Murali Krishna K, Ahmed R. Cutting edge: naive T cells masquerading as memory cells. J Immunol. 2000;165:1733-1737[Abstract/Free Full Text]. 16. Masuko K, Kato S, Hagihara M, et al. Stable clonal expansion of T cells induced by bone marrow transplantation. Blood. 1996;87:789-799[Abstract/Free Full Text]. 17. Hingorani R, Choi IH, Akolkar P, et al. Clonal predominance of T cell receptors within the CD8+ CD45RO+ subset in normal human subjects. J Immunol. 1993;151:5762-5769[Abstract]. 18. Mackall CL, Granger L, Sheard MA, et al. T-cell regeneration after bone marrow transplantation: differential CD45 isoform expression on thymic-derived versus thymic-independent progeny. Blood. 1993;82:2585-2594[Abstract/Free Full Text]. 19. Weinberg K, Blazar BR, Wagner JE, et al. Factors affecting thymic function after allogeneic hematopoietic stem cell transplantation. Blood. 2001;97:1458-1466[Abstract/Free Full Text]. 20. Bolotin E, Smorgorzewska M, Smith S, et al. Enhancement of thymopoiesis after bone marrow transplant by in vivo interleukin-7. Blood. 1996;88:1887-1897[Abstract/Free Full Text]. 21. Mackall CL, Fry TJ, Bare C, et al. IL-7 increases both thymic-dependent and thymic-independent T-cell regeneration after bone marrow transplantation. Blood. 2001;97:1491-1497[Abstract/Free Full Text]. 22. Bader P, Holle W, Klingebiel T, et al. Mixed hematopoietic chimerism after allogeneic bone marrow transplantation: the impact of quantitative PCR analysis for prediction of relapse and graft rejection in children. Bone Marrow Transplant. 1997;19:697-702[CrossRef][Medline] [Order article via Infotrieve]. © 2002 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: J.-F. Poulin, M. Sylvestre, P. Champagne, M.-L. Dion, N. Kettaf, A. Dumont, M. Lainesse, P. Fontaine, D.-C. Roy, C. Perreault, et al. Evidence for adequate thymic function but impaired naive T-cell survival following allogeneic hematopoietic stem cell transplantation in the absence of chronic graft-versus-host disease Blood, December 15, 2003; 102(13): 4600 - 4607. [Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) All Versions of this Article: 2001-11-0005v1 100/5/1915    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Eyrich, M. Articles by Schlegel, P. 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	Copyright © 2002 by American Society of Hematology         Online ISSN: 1528-0020