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Prepublished online as a Blood First Edition Paper on August 8, 2002; DOI 10.1182/blood-2002-03-0976.
IMMUNOBIOLOGY
From the Departments of Clinical Medicine and
Experimental Medicine and Pathology, University "La Sapienza," and
Molecular Oncogenesis Laboratory, Istituto Dermopatico dell'
Immacolata, Rome, Italy; San Raffaele Telethon Institute
for Gene Therapy, Milan, Italy; and Department of
Biomedical Sciences, University of Modena and Reggio Emilia,
Italy.
Ataxia telangiectasia (A-T), a genetic disorder caused by the
homozygous mutation of the ATM gene, frequently
associates with variable degrees of cellular and humoral
immunodeficiency. However, the immune defects occurring in patients
with A-T are still poorly characterized. Here we show that the T-cell
receptor (TCR) variable Ataxia telangiectasia (A-T) is an autosomal
recessive multisystem disorder characterized by cerebellar ataxia,
conjunctival and cutaneous telangiectasias, immune deficiency,
chromosome aberrations, radiation hypersensitivity, and a high
incidence of tumors, mainly of lymphoid origin.1 Immune
deficiency of A-T patients is heterogeneous and involves both cellular
and humoral responses.2,3 Typically, humoral
immunodeficiency consists of the reduction of IgG2, IgA, and IgE with
normal to increased IgM. The thymus is hypoplastic, and circulating T
cells are usually decreased with a predominance of those expressing the
The gene responsible for A-T, which has been identified by positional
cloning and denoted ATM (ataxia-telangiectasia mutated), encodes a protein with phosphatidylinositol 3-kinase (PI3K) signature at the carboxyl terminus.5 The PI3K family includes
several proteins involved in cell cycle control, telomere maintenance, and repair of DNA double-strand breaks (DSBs).6 ATM is
localized mostly to the nucleus and associates with DNA, with
particular affinity for DNA ends. In the presence of DNA DSB damage,
ATM phosphorylates a variety of protein targets, including p53, cAbl, replication protein A (RPA), Chk1, and Chk2, and activates multiple signal transduction pathways.7
Mice with disrupted ATM gene (ATM The mechanism(s) of the immunodeficiency consequent to inactivation of
the ATM gene have not yet been fully
elucidated.11 Chromosomal translocations involving
immunoglobulin and TCR genes at 7p14, 7q35, 14q11.2, and 14q32 are
commonly observed in neoplastic and nonneoplastic lymphocytes of
patients with A-T. This suggests that illegitimate joining during V(D)J
recombination might underlie both impaired lymphocyte development and
enhanced lymphomagenesis.12-14 The finding that T-cell
number and function can be rescued in ATM To gain further insight into the mechanism(s) causing immunodeficiency
in A-T, we investigated the repertoires of TCR Patients
The patients with partial DiGeorge syndrome and typical 22q11.2
deletion have been described in a previous publication.22
All experiments were done using anticoagulant-treated peripheral blood
obtained by venipuncture. This study was conducted according to the
good clinical practice guidelines of the Italian Ministry of Health.
Informed consent was obtained from all patients or parents.
Flow cytometric analysis of CD4+ and CD8+
T-cell subsets and TCRBV repertoire
Single-cell analysis of cytokine production Analysis of cytokine production at the single-cell level was performed as previously described.23 Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from heparinized venous blood by Ficoll-Isopaque (Lymphoprep-Nycomed, Oslo, Norway) gradient centrifugation, counted, and resuspended at 1 × 106 cells/mL in RPMI 1640 medium (Gibco Laboratories, Grand Island, NY) supplemented with 10% FBS (Sigma, St Louis, MO), 2 mM glutamine (Sigma), and 50 µg/mL gentamycin (Life Technologies, Gaithersburg, MD). Cells were then stimulated for 16 hours with 1 µg/mL ionomycin (Sigma) and 25 ng/mL phorbol myristate acetate (Sigma) in the presence of 10 µg/mL brefeldin A to inhibit cytokine secretion. After a wash in PBS, cells were fixed with 4% paraformaldehyde by incubation for 5 minutes at room temperature, permeabilized with FACS permeabilizing solution (Becton Dickinson, Immunocytometry Systems) for 10 minutes, washed, and stained. The following cytokine-specific mAbs were used: FITC-labeled anti-human interferon (anti-hIFN-; IgG2b), FITC-labeled
anti-human interleukin 2 (anti-hIL-2; IgG1), and PE-labeled
anti-hIL-4 (IgG1). Surface phenotyping was performed with anti-CD4 APC
and anti-CD8 PerCP. All the mAbs were purchased from Becton Dickinson
Immunocytometry Systems. After staining cells were washed once in PBS
containing 10% FBS and analyzed on a FACSCalibur cytofluorometer
(Becton Dickinson, Immunocytometry Systems) using the Cell Quest
software. To determine the frequency of cytokine-producing T cells,
total lymphocytes were first gated by forward and side scatter and then
additionally gated for CD4 or CD8 expression; 20 000 gated events were
collected for each sample. Appropriate isotypic negative controls were
run in parallel.
Molecular studies CD4+ and CD8+ T cells were separated by using CD4 and CD8 MicroBeads and MACS columns according to the manufacturer's protocols (Miltenyi Biotec, Bergisch Gladbach, Germany). Total mRNA was extracted directly from 106 to 107 bead-coated cells using Trizol-LS Reagent (Gibco-BRL, Grand Island, NY) and Micro-carrier (Molecular Research Center, Cincinnati, OH) and precipitated with isopropyl alcohol. The pelleted RNA was resuspended in diethyl-pyrocarbonate-treated water and the poly-(A)+ portion of total RNA was converted into cDNA using 2.5 µM oligo-deoxythymidine (dT) as primer for reverse transcription (RT), 50 mM KCl, 10 mM Tris (tris(hydroxymethyl)aminomethane)-HCl, 5 mM MgCl2, 1 mM of each deoxyribonucleoside triphosphates (dNTPs), 1 U/µL RNase inhibitor, and 2.5 U/µL murine leukemia virus (MULV) reverse transcriptase (Applied Biosystems, Foster City, CA).To analyze the TCRBV transcript size patterns cDNA samples were amplified by using a TCRB C1/C2-specific primer (CGG GCT GCT CCT TGA GGG GCT GCG) and a set of 24 TCRBV-specific primers (BV 1, 2, 3, 4, 5.1, 5.3, 6.1, 6.2, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 24).24 Briefly, 2 µL of the RT product was brought to a final reaction volume of 50 µL containing 50 mM KCl, 10 mM Tris-HCl 1,5 mM MgCl2, 0,2 mM of each dNTPs, 25 pmol of each oligonucleotide, and 2 U Taq DNA polymerase (AmpliTaq; Applied Biosystems). After an initial denaturation step of 3 minutes at 95°C the reactions were subjected to 35 cycles of polymerase chain reaction (PCR; 30 seconds at 94°C, 30 seconds at 60°C, 30 seconds at 72°C) followed by a final elongation step for 10 minutes at 72°C. Aliquots of the unlabeled PCR products were then labeled by 10 cycles of elongation in a 10 µL "run-off" reaction with the FAM TCRBC primer (CTG CAC CTC CTT CCC ATT) mixed with deionized formamide and TAMRA 500 size standard (Applied Biosystems). Finally, run-off products were electrophoresed for 24 minutes on a 310 ABI PRISM automated sequencer by using a 47-cm capillary and POP-4 polymer. The third complementarity-determining region (CDR3) profile was then analyzed with the Genescan software (Applied Biosystems). For IgH CDR3 spectratyping, amplifications were done with oligonucleotide primers specific for constant µ chain (Cµ) and for a conserved sequence in the human VH framework region 3.25 Analysis of the level of perturbation of TCRBV repertoire of A-T patients was performed according to Gorochov et al26 with minor modifications. Briefly, the CDR3 length profiles (spectratypes) were first translated into probability distributions as function of the area under the profile for each CDR3 length. A control profile, representing the nonperturbed repertoire, was determined for each BV by calculating the average distribution of the corresponding CD4 and CD8 profiles from 5 healthy blood donors. The extent of perturbation for each CDR3 fragment was then calculated by the difference between the sample's distribution and the control's distribution. Finally, the TCR repertoire perturbation per BV family was defined as the sum of the absolute values of the differences between each sample's CDR3 length and the corresponding control distribution. Values of BV perturbation greater than the sum of the SDs calculated in healthy blood donors for each CDR3 profile were considered abnormal. For CDR3 sequencing, amplification products were inserted into a plasmid vector (pCRII-TOPO; Invitrogen, Carlsbad, CA), cloned in Escherichia coli and sequenced onto an Applied Biosystems Sequencer Model 377-96. Sequence similarities were identified using the multiple sequence alignment application, Align X, of the Vector NTI Suite 6.0 (Informax, North Bethesda, MD) based on the CLUSTAL W algorithm.27 The identification of the V(D)J junctions was performed comparing the sequences with those reported in IMGT, the International ImMunoGeneTics database http://imgt.cines.fr:8104 (Initiator and coordinator: Marie-Paule Lefranc, Montpellier, France).28 For measuring TRECs,
T-cell phenotypes In most of the patients investigated the peripheral distribution of CD4+ T cells was lower than observed in age-matched controls, whereas that of CD8+ T cells generally fell within normal range (Table 1). The large majority of T cells expressed CD95 (for CD4+: 86% ± 18% versus 39% ± 11% in healthy controls, P < .001; for CD8+: 87% ± 18% versus 36% ± 12% in controls), denoting an activated phenotype. The distribution of naive and memory T cells was investigated by the differential expression of CD45 isoforms and of CD62L. All A-T patients, with the exception of patient no. 7, showed a marked predominance of T cells with a memory phenotype (CD45RA CD62L,
CD45RA+CD62L,
CD45RACD62L+; Table 1). This was seen in
association with a corresponding reduction in the frequency of naive T
cells, particularly pronounced within the CD4 subset.
The expression of CD62L, a lymph node homing receptor, may distinguish
"central memory" T cells, which are CD62L+ and produce
mainly IL-2, from terminally differentiated "effector memory" T
cells, which are CD62L The pattern of activation-induced cytokine production by patient's T
cells (Table 1) was consistent with their predominant memory phenotype.
In fact, compared to healthy controls, there was a significant increase
of cells polarized toward the production of IFN- Taken together, our findings indicate that most circulating T cells of A-T patients are memory cells, predominantly with the features of terminally differentiated effectors, whereas naive T cells are very scarce. Thymic function During V(D)J T-cell receptor rearrangement, DNA extrachromosomal excision products (also known as TRECs) are generated. These products are not replicated during mitosis so that their number decreases with each round of cell division. For this reason TRECs are usually used as a marker of recent thymic emigrants (RTEs).29 Based on these notions, we investigated whether the defective numbers of naive T cells we found in A-T patients could be attributed to low thymic output by evaluating the levels of TRECs in separated CD4+ and CD8+ T cells.All A-T patients displayed extremely low levels of TRECs in comparison with age-matched healthy individuals (Table 1) and even in comparison with 4 patients with DiGeorge syndrome, an immunodeficiency caused by thymic hypoplasia, who had, respectively, 18.5, 21.7, 78, and 68 copies/100 ng DNA in CD4 cells, and 49.8, 104, 46, and 27 copies/100 ng DNA in CD8 cells. Analysis of the TCRBV repertoire by mAbs Anomalies in the relative TCRBV usage were investigated using a panel of BV subfamily-specific mAbs covering approximately 60% to 70% of T cells expressing TCR  in healthy individuals (Table 2).
Among the 24 TCRBV genes investigated, significant expansions (ie,
above the highest values observed in healthy subjects) were observed in
9.5% of V To investigate the capacity of A-T patients to generate a normally wide
TCRBV repertoire, we looked for "holes" in the expression of V Molecular analysis of the TCRB and IgH repertoires Heterogeneity of the TCRB repertoire was further investigated by CDR3 "spectratyping," that is, by the quantitative analysis of CDR3s with different sizes generated by the random insertion/deletion of nucleotides during V(D)J rearrangement.24 A normal, polyclonal repertoire results in a histogram with a gaussianlike distribution of CDR3 lengths, whereas abnormal patterns display one or more predominant fragments outside the peak of median length (Figure 1). Mathematical analysis of the deviation of patients' histograms from the normal distribution revealed that all A-T patients had significantly altered patterns in most V genes examined (Figure 2A-I).
These alterations are indicative of V repertoires restricted by
diffuse clonal expansions. As expected, the profiles of CDR3 size
distribution were more perturbed within the CD8+ subset,
with oligoclonal peaks found in each BV family investigated (Figure 1).
In fact, disruption of the gaussianlike CDR3 profile of some BV
families is often observed in CD8+ lymphocytes of healthy
individuals. In healthy subjects these idiosyncratic peaks are constant
over time and, therefore, do not necessarily represent ongoing immune
responses. However, in A-T patients perturbations of the TCRBV
repertoire of CD8+ cells were much more prominent and
systematic than in healthy controls, indicating that they were most
likely related to the underlying immunologic disorder.
The CDR3 spectratypes obtained by amplification of IgH-specific
sequences were also altered in most patients with A-T (Figure 2J),
suggesting the presence of B-cell oligoclones. B-cell oligoclonality in
A-T could be due either to restricted central generation or to
dysregulated peripheral expansion caused by defects in regulatory T
cells. To discriminate between these 2 possibilities, we examined patients with DiGeorge syndrome, whose immunodeficiency is solely attributable to a thymic defect, and observed a normal distribution of
IgH CDR3 spectratypes (not shown). This suggests that decreased diversity of the B-cell repertoire in A-T patients is due to an intrinsic developmental defect, in agreement with the observation that
ATM
To gain further information on the TCRB and IgH repertoires as well as on the V(D)J joining process in A-T, we sequenced over 250 CDR3 regions from selected TCRBV and IgH amplification products from 3 patients (nos. 5, 8, and 9), and an equivalent number from a healthy donor. The A-T patients displayed an oligoclonal repertoire of productively
rearranged TCRBV genes (Figure 3). The
most restricted repertoire was observed within CD4 V
The IgH coding joints were analyzed in 2 A-T patients (nos. 8 and 9) and in 1 healthy subject. Patient no. 8 had a relatively normal repertoire, with 91% variability compared to 98% in a healthy subject (data not shown). By contrast, patient no. 9 had a more restricted pattern displaying 62% variability (Figure 3). Nearly all patients' TCRBV and IgH V(D)J coding joints sequenced were productively rearranged. We observed only occasional abnormally assembled coding joints (data not shown) that are being further investigated. The TCRB and IgH CDR3 average lengths were similar in A-T patients (6-13 amino acids) and in healthy subjects (6-15 amino acids), and corresponded to previously reported estimates.35-37 Also, we did not notice any preferential usage of TCRB joining genes38 in A-T patients.
Our present findings highlight several aspects of the
immunodeficiency associated with A-T. We found that the TCRBV
repertoire of A-T patients is skewed by either underusage or
oligoclonal expansion of most V The putative recombination defect of A-T does not appear to involve
coding joint formation, because we found perfectly normal CDR3
sequences derived from endogenous TCR A striking finding in our study was that the TCRBV repertoire of A-T patients is skewed by diffuse oligoclonal expansions. Severe restriction of the TCR repertoire has been observed in patients with recombination defects due to RAG mutations,39,40 in patients with complete DiGeorge syndrome who have extremely low levels of T cells because of the absence of thymic epithelium,45 and in patients infected with HIV with low CD4 cell counts.26 However, patients with partial DiGeorge syndrome (Pierdominici et al46; A.G., M.P., unpublished data, 2002), or HIV-infected patients at early stages of the disease who have a moderate reduction of CD4 T cells,26,47,48 have substantially normal CD4 TCR repertoires. Thus, it is likely that restriction of the TCR repertoire in A-T depends more on constraints of TCR generation rather than on a generalized decrease of thymopoiesis. Peripheral T-cell oligoclones probably originate from the peripheral expansion of the relatively few cells that achieve functional TCR rearrangements.39 The possibility that these expansions were due to chronic infections is unlikely, because antigenic stimulation predominantly drives the expansion of CD8 cells,31,32 whereas oligoclones predominated in CD4 cells of our A-T patients. Furthermore, oligoclonal expansions are not observed in chronically infected patients with other immune defects such as major histocompatibility complex (MHC) class II deficiency.49 The phenotypic and functional data discussed below argue in favor of this hypothesis. We found that patients' circulating T cells are predominantly
represented by terminally differentiated effector memory cells. Several
studies have suggested that T and B lymphocytes from A-T patients have
subtle defects in intracellular signal transduction,18-20 suggesting that abnormal responsiveness to antigenic stimulation might
account, at least in part, for immunodeficiency. Our finding that
patients' T cells have the phenotype and the polarized pattern of
cytokine production typical for effector memory cells strongly argues
against this interpretation, because it implies that these cells must
have responded properly to in vivo antigenic stimulations to become
terminally differentiated effectors. Therefore, T-cell immunodeficiency
in A-T is more likely to depend on a developmental defect rather
than on defects of T-cell activation. This hypothesis is consistent
with the low thymic output suggested by the dramatic reduction of TRECs
in our patients. Although a decrease of TRECs might be the consequence
of peripheral T-cell activation rather than of low thymic
output,50 our finding is more likely to reflect the thymic
hypoplasia typical for A-T patients and for ATM B-cell development has been investigated less thoroughly in
ATM
We are extremely grateful to A. M. R. Taylor for critical reading of the manuscript and to Grazia Andolfi for excellent technical assistance.
Submitted March 29, 2002; accepted July 8, 2002.
Prepublished online as Blood First Edition Paper, August 8, 2002; DOI 10.1182/blood-2002-03-0976.
Supported in part by the Fondazione Istituto Pasteur-Fondazione Cenci Bolognetti at the University of Rome "La Sapienza," by the Italian Ministry of Health Progetto Finalizzato 2000, by the European Community Grant QLG2-CT-1999-00786, by Telethon grants D.102 and and E.0764, and by the Associazione Italiana Ricerca sul Cancro.
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: Antonello Giovannetti, Department of Clinical Medicine, University of Rome "La Sapienza," Viale dell'Università 37, 00185 Rome, Italy; e-mail: antonello.giovannetti{at}uniroma1.it.
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  J. Bagley, G. Singh, and J. Iacomini Regulation of Oxidative Stress Responses by Ataxia-Telangiectasia Mutated Is Required for T Cell Proliferation J. Immunol., April 15, 2007; 178(8): 4757 - 4763. [Abstract] [Full Text] [PDF]
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A. Giovannetti, M. Pierdominici, F. Mazzetta, M. Marziali, C. Renzi, A. M. Mileo, M. De Felice, B. Mora, A. Esposito, R. Carello, et al. Unravelling the Complexity of T Cell Abnormalities in Common Variable Immunodeficiency J. Immunol., March 15, 2007; 178(6): 3932 - 3943. [Abstract] [Full Text] [PDF]
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 I. R. Matei, R. A. Gladdy, L. M. J. Nutter, A. Canty, C. J. Guidos, and J. S. Danska ATM deficiency disrupts Tcra locus integrity and the maturation of CD4+CD8+ thymocytes Blood, March 1, 2007; 109(5): 1887 - 1896. [Abstract] [Full Text] [PDF]
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A. Allam and D. Kabelitz TCR trans-Rearrangements: Biological Significance in Antigen Recognition vs the Role as Lymphoma Biomarker J. Immunol., May 15, 2006; 176(10): 5707 - 5712. [Abstract] [Full Text] [PDF]
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M. Carbonari, E. Caprini, T. Tedesco, F. Mazzetta, V. Tocco, M. Casato, G. Russo, and M. Fiorilli Hepatitis C Virus Drives the Unconstrained Monoclonal Expansion of VH1-69-Expressing Memory B Cells in Type II Cryoglobulinemia: A Model of Infection-Driven Lymphomagenesis J. Immunol., May 15, 2005; 174(10): 6532 - 6539. [Abstract] [Full Text] [PDF]
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 J. M. Lumsden, T. McCarty, L. K. Petiniot, R. Shen, C. Barlow, T. A. Wynn, H. C. Morse III, P. J. Gearhart, A. Wynshaw-Boris, E. E. Max, et al. Immunoglobulin Class Switch Recombination Is Impaired in Atm-deficient Mice J. Exp. Med., November 1, 2004; 200(9): 1111 - 1121. [Abstract] [Full Text] [PDF]
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J. Bagley, M. L. Cortes, X. O. Breakefield, and J. Iacomini Bone marrow transplantation restores immune system function and prevents lymphoma in Atm-deficient mice Blood, July 15, 2004; 104(2): 572 - 578. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
T-cell receptor (TCR) relative to those expressing the 
indicates, abnormal pattern in CD4 cells; 
, abnormal pattern in CD8
cells; 
plus *, normal pattern. Panel J represents IgH data in
patients nos. 1 to 9; 