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Characterization of the T Cell Receptor Repertoire of Neonatal T Cells by RT-PCR and Single Strand Conformation Polymorphism Analysis

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Characterization of the T Cell Receptor Repertoire of Neonatal T Cells by RT-PCR and Single Strand Conformation Polymorphism Analysis Bone Marrow Transplantation (2000) 26, 83–89 2000 Macmillan Publishers Ltd All rights reserved 0268–3369/00 $15.00 www.nature.com/bmt Characterization of the T cell receptor repertoire of neonatal T cells by RT-PCR and single strand conformation polymorphism analysis E Alfani1, AR Migliaccio2, M Sanchez1, AM Passarelli3 and G Migliaccio1 1Laboratory of Cell Biology and 2Clinical Biochemistry, Istituto Superiore di Sanita`, Rome; and 3Ospedale Civile Tivoli, Tivoli, Italy Summary: by other molecular mechanisms which include imprecise joining of V(D)J recombination,6 N-region diversification We have analyzed by reverse transcriptase-polymerase (random addition of nucleotides by terminal deoxy-ribonu- chain reaction (RT-PCR) the individual non-germ line cleotidyl transferase)7,8 and insertions of palindromic configurations of the T cell receptor (TCR) V␤ chains nucleotides9 at specific points of the VD, DJ and VJ junc- expressed by T cells from eight individual cord blood tions. As a result of allele exclusion,10 each T cell clone specimens. cDNA from each cord blood was amplified expresses one and only one specifically rearranged TCR using a common primer coupled with a primer specific V␤ chain. Therefore, the number of TCR V␤ chain for each of 22 variable elements of the V␤ chain family rearrangements expressed by a given population correlates and the amplified fragments were separated under high with its T cell clonality. resolution conditions. With cDNA from adult blood (as Cord blood (CB) has been proven to be a good source a control), all of the TCR chains were amplified as a of stem/progenitor cells for related and unrelated trans- smear consistent with the extensive polyclonality of plants.11,12 Successful engraftment with low graft-versus- adult T cells. In contrast, a heterogeneous pattern of host disease (GVHD) has been observed even in the case amplification was observed with cDNAs from cord of unrelated poorly matched transplants.13,14 The reduced blood: only 26.7 ± 21.9% of the 22 V␤ chains analyzed GVHD severity observed in allogeneic CB transplantation were amplified as a smear. The majority of them were is explained by the low immunologic reactivity of the neo- amplified as a discrete number of bands (up to 10) (in natal T lymphocytes.15–17 The reasons for such immaturity 68.2 ± 18.7% of samples) and some of them as a single is not yet fully understood but it is possible that it is fragment (4.0 ± 7.8%). Only one of the eight samples mediated by cooperation between different mechanisms. analyzed expressed the majority (72.7%) of its V␤ For example, some studies have reported that CB contains chains as a smear, consistent with an adult-like TCR reduced numbers of CD45RO+ memory cells,18 while repertoire. In conclusion, cord blood expressed, on aver- others have suggested an intrinsic immaturity of CB T cells, age, a less complex TCR repertoire than adult blood. independent of the CD45 isoform they express.19–21 Other Bone Marrow Transplantation (2000) 26, 83–89. authors have observed that neonatal T lymphocytes do pro- Keywords: cord blood; T cell receptor; single strand liferate in response to several antigens, suggesting that anti- conformation polymorphism; cord blood transplantation; gen primed T cells are present in neonatal blood,22–25 and graft-versus-host disease that it is a reduced functional capacity of accessory cells, such as APC, which underlies the ‘immaturity’ of the neonatal immune system.26 During human ontogeny, complete functional TCR V␤ T lymphocytes are cells produced in the thymus that, once chain transcripts begin to be expressed by the thymocytes activated, are responsible for the humoral and cellular in the thymus at 15 weeks of gestation,27–29 and almost all immune responses. The process of T cell activation is the different TCR V␤ genes are expressed by the 17th mediated by the T cell receptor (TCR), a membrane-bound week.6 Since the molecular mechanisms which determine ␣ ␤ heterodimer composed of an and a chain, that recog- the non-germ line V␤ gene diversity are not fully active nizes antigens once coupled with the major histocompat- until the 32nd week,29 it is debated whether the T cells ibility complex expressed by antigen presenting cells circulating in CB are as polyclonal as those circulating in ␤ (APC). During the process of T cell differentiation, the adult blood. Schelonka et al30 have observed, in 35 separate chains undergo rearrangements through somatic recombi- CB samples analyzed by spectra-typing, a highly polyclonal nation among multiple coding segments, namely the vari- TCR repertoire before the 28th week of gestation and oligo- 1,2 able (V), diversity (D), joining (J) and constant (C) genes. clonal T cell expansion at 29th–33rd weeks. This oligoclon- ␤ At present, more than 65 different V genes, a quarter of ality would indicate a reduction in the complexity of the which are represented by pseudogenes, have been ident- circulating T cell repertoire around birth. In contrast with 3–5 ified. The V(D)J junctional diversity is further increased these observations, Garderet et al,31 by analyzing the lengths of the complementarity determining region 3 (CDR Correspondence: Dr G Migliaccio, Laboratory of Cell Biology, Istituto 3) by immunoscopy (a computer-based program developed Superiore di Sanita`, Viale Regina Elena 299, 00161 Rome, Italy to analyze the numbers of fluorescent RT-PCR products Received 19 November 1999; accepted 7 March 2000 obtained with a given pair of primers and separated on a Reduced T cell receptor repertoire in cord blood E Alfani et al 84 sequencing gel) in nine separate CB, have reported that CB RNA extraction and cDNA synthesis T cells express a mature polyclonal TCR repertoire like adult T cells. The discrepancy between these two sets of Nucleated cells were counted and washed twice in HBSS. data could be due to technical limitations. In fact, both After centrifugation, the cell pellet was lysed with guani- spectra-typing and immunoscopy do not discriminate dine isothiocyanate and mRNA extracted with oligo(dT) between V␤ chain rearrangements identical in length but spun columns (Quick Prep Micro mRNA Purification kit; different in sequence. Amersham Pharmacia Biotech) as described by the manu- To clarify whether CB T cells express a reduced com- facturer. RNA was retro-transcribed with Superscript II plexity of the TCR repertoire, the non-germ line configur- reverse transcriptase and oligo(dT) primers (Gibco-BRL, ations of 22 (out of the 25 described) V␤ chain families Grand Island, NY, USA). expressed by eight CB specimens (and four adult PB as control) were compared by the very sensitive method Polymerase chain reaction analysis of the expression of recently described,32,33 which combines reverse tran- the non-germ line configurations of the TCR V␤ chains scriptase-polymerase chain reaction (RT-PCR) amplifi- cation of the V␤ chain transcripts with the separation of Individual non-germ line configurations of the TCR V␤ the amplified products by single strand conformation poly- elements were amplified with a common primer coupled morphism (SSCP) analysis. This technique, although not as with a primer specific for each of the 22 variable elements sensitive as heteroduplex analysis,34 allows discrimination of the V␤ chain as described.35,36 cDNA obtained from pur- of V␤ products differing by a single nucleotide substi- ified mRNA extracted from an equivalent number of cells tution.33 The results obtained indicate that CB expresses, (Ӎ1 × 106) was used in each reaction. Hypoxanthine phos- on average, a reduced T cell clonality, although one out of phoribosyl transferase (HPRT) was also amplified as a con- the eight specimens analyzed had an adult-like repertoire. trol for the quality and quantity of cDNA. cDNA was We conclude that the immaturity of the neonatal T cell sys- amplified with Pfu DNA polymerase (0.5 U/reaction, Strat- tem includes, at least in some CB specimens, a reduced agene USA, La Jolla, CA, USA) for 35 cycles in a PTC- number of T cell clones. 100 Thermocycler (MJ Research, Watertown, MA, USA). The amplified products were denatured and separated on a 5% acrylamide 10% glycerol non-denaturing gel.33 The gel was blotted on to Hybond N+ membrane (Amersham Phar- Materials and methods macia Biotech). The specificity of the amplification was confirmed by probing with end-labelled oligonucleotides (␥-32P-ATP, specific activity = 3000 Ci/mMol, Du Pont, Specimen collection and cell purification Firenze, Italy and T4 polynucleotide kinase end-labelling kit, Gibco BRL) specific for the constant portion of the Umbilical cord blood was obtained at the time of delivery V␤ elements.37 After hybridization, the membranes were from uncomplicated pregnancies with previous written washed twice under stringent conditions and exposed to X- informed consent from the mother. Peripheral blood (PB) ray films (Eastman Kodak Company, Rochester, NY, was obtained from healthy adult donors through the collab- USA). All the procedures were conducted according to oration of the Italian Red Cross. The human subject proto- standard protocols.38 col was approved with the premise that no personal data would be linked with the samples analyzed. Mononuclear cells (MC) were isolated from a total of 11 cord blood (CB) Statistical analysis and four PB specimens (all fresh) by density-cut separation Analysis of variance (ANOVA) was performed with the ␳ Ͻ ( 1.077) (Ficoll-Paque; Amersham Pharmacia Biotech, Origin 4.10 software for Windows (Microcal Software, Uppsala, Sweden). CBMC and PBMC were either pro- Northampton, MA, USA). cessed directly or cryopreserved with dimethyl sulfoxide (DMSO 10% v/v, in Hanks’s balanced salt solution, HBSS, Sigma, St Louis, MO, USA).35 Results V␤ gene rearrangements expressed by T cells from cord Cell immune-phenotyping blood and adult pheripheral blood Cells were washed twice in HBBS and incubated (3– The analysis by RT-PCR and SSCP of the TCR V␤ chain 5 × 105 cells per tube) at 4°C for 30 min with monoclonal repertoire expressed by adult PB and CB specimens is antibodies against CD4, CD7, CD8, CD45, CD45RA and presented in Figure 1 and in Table 1.
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