|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 10 (November 15), 1999:
pp. 3468-3478
Intrathymic Restriction and Peripheral Expansion of the T-Cell
Repertoire in Omenn Syndrome
By
Simona Signorini,
Luisa Imberti,
Silvia Pirovano,
Anna Villa,
Fabio Facchetti,
Marco Ungari,
Fabio Bozzi,
Alberto Albertini,
Alberto
G. Ugazio,
Paolo Vezzoni, and
Luigi D. Notarangelo
From the Terzo Servizio Analisi, Spedali Civili, the Institute of
Chemistry, the Department of Pathology, and the Istituto di Medicina
Molecolare "Angelo Nocivelli," Department of Pediatrics,
University of Brescia, Brescia, Italy; and the Department of Human
Genome and Multifactorial Disease Research, I.T.B.A. CNR, Segrate (MI),
Italy.
 |
ABSTRACT |
Mutations in the human RAG genes that impair, but do not
abolish, recombination activity lead to Omenn syndrome, a severe primary immune deficiency that is associated with clinical and pathological features of graft-versus-host disease and oligoclonal expansion of activated, autologous T cells. We have analyzed the mechanisms accounting for peripheral oligoclonality of the T-cell repertoire. Predominance of few T-cell receptor clonotypes (both within
TCRAB- and within TCRGD-expressing lymphocytes) is already detectable in the thymus and is further selected for in the
periphery, with a different distribution of clonotypes in different
tissues. These data indicate that oligoclonality of the T-cell
repertoire in Omenn syndrome is due both to intrathymic restriction and
to peripheral expansion. Moreover, the RAG genes defect that
causes Omenn syndrome directly affects early stages of V(D)J
recombination, but does not alter the process of double-strand-break
DNA repair, including N and P nucleotide insertion.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
OMENN SYNDROME (OS) is a rare, autosomal
recessive combined immune deficiency characterized by diffuse
erythrodermia, lymphadenopathy, hepato-splenomegaly, protracted
diarrhea, failure to thrive, hypereosinophilia, and
hypogammaglobulinemia, but elevated serum IgE.1-3 Unless
treated with bone marrow transplantation, OS patients die of
overwhelming infections and severe metabolic disturbances within the
first months of life.4
A peculiar immunological phenotype has been demonstrated in OS. B cells
are usually absent both in peripheral blood and in lymphoid tissues
from these patients.2-9 In contrast, although lymph nodes
and thymus show depletion of lymphoid cells, a variable number of
autologous, activated, poorly functional and oligoclonal T cells, with
a skewed Th2 profile,7,10-12 are present in peripheral blood and infiltrate the skin, gut, liver, and
spleen.6,9,13-15
We have recently demonstrated that the disease is due to mutations in
the genes that encode for RAG1 and RAG2, two key lymphoid-specific proteins that are essential for V(D)J recombination.16
Whereas null mutations that completely eliminate the recombination
activity of these proteins cause a complete block of T- and B-cell
development and lead to severe combined immune deficiency with absence
of mature T and B lymphocytes (T B
SCID),17 the RAG gene mutations identified in OS
patients allow the recombination machinery to rearrange, albeit with
reduced efficiency, T-cell receptor (TCR) gene segments.16
Although the identification of RAG1 and RAG2 mutations
represents a first step in understanding the pathogenesis of OS,
several questions remain. In particular, we and others have shown that the peripheral T-cell repertoire in OS is highly
restricted,6,13-16 but the mechanisms that account for this
phenomenon are still poorly defined. The selective accumulation of a
few T-cell clones in the periphery might reflect the preferential
expression of only a few TCR specificities already in the thymus (due
to the RAGs defect); alternatively, it might be the consequence
of an (auto)antigen-driven peripheral expansion of a few clones out of
an otherwise diversified pool of T cells generated in the thymus.
The availability of several tissues (including thymus) obtained from an
OS patient gave us the opportunity to discriminate between these
possibilities by comparing the pattern of V(D)J recombination in
thymocytes and in T lymphocytes that infiltrate different peripheral
target tissues. Furthermore, sequence analysis of the productively
rearranged TCR gene segments in a series of patients with molecularly
characterized OS allowed us to investigate if the RAG gene
defects that account for OS result in peculiar constraints over the
process of V(D)J recombination and generation of TCR specificities.
| |
PATIENT AND METHODS |
Patient.
The patient, a female infant, was the first child of a couple of
consanguineous parents. She developed diffuse erythrodermia at 2 weeks
of age, followed by persistent cough, protracted diarrhea, and failure
to thrive. At 1 month of age, laboratory examinations performed during
hospitalization showed hypoproteinemia and low serum IgG (205 mg/dL)
and IgA (8 mg/dL), but increased serum IgE (9,100 kU/L) and moderate
thrombocytopenia (92 × 109/L). Total lymphocytes were
8,600/µL, with a predominance of TCRGD (32%) versus TCRAB (11%) T
cells. Other subsets were as follows: CD3, 43%; CD4, 11% (CD45RA,
<1%; CD45R0, 11%); CD8, 17%; CD19, <1%; CD16, 46%; CD25, 12%;
and CD3+DR+, 38%. In vitro proliferative
response to mitogens (in counts per minute [cpm] × 103) was markedly decreased: phytohemagglutinin, 7.5 (v 108.3 in an age-matched control); anti-CD3 monoclonal
antibody, 5.5 (v 85.8); and phorbol myristate acetate plus
ionomycin, 6.2 (v 45.9). Based on these findings, a diagnosis
of OS was established. Despite isolation in a protected environment and
a supply of intravenous Igs, antibiotics, acyclovir, and albumin, the
conditions of the infant worsened, and she died of heart failure and
respiratory distress at 3 months of age. Postmortem examination showed
myocarditis and pneumonia.
Mutation analysis at the RAG loci.
The RAG1 and RAG2 coding sequences were amplified from
genomic DNA. Primers were designed for the amplification of the
RAG genes based on their sequence, as previously
described16 (RAG1 accession no. M29474 and
RAG2 accession no. M94633). The RAG1 gene was amplified
in 2 segments (nt 94-1852 and nt 1653-3309), and RAG2 was
amplified in 1 segment (1201-2922). Sequencing was performed either
directly on the polymerase chain reaction (PCR) product purified from
the gel and sequenced using Thermosequenase Kit (Amersham, Amersham,
UK) or on PCR products cloned in TA vector (Invitrogen,
San Diego, CA). Sequencing was performed by the dideoxynucleotide chain
termination method using the Sequenase.
Tissues and immunohistochemistry.
Tissue specimens were represented by an inguinal lymph node and a skin
biopsy ob- tained at the age of 3 months and various organs that were
removed at the time of postmortem examination. All specimens were fixed
in formalin and embedded in paraffin; in addition, a small
fragment was also frozen in liquid nitrogen and stored at
 80°C.
Immunohistochemistry was performed with an indirect streptavidin-biotin
complex immunoperoxidase technique on cryostat sections that were
air-dried for 18 hours after cutting and fixed in acetone for 10 minutes before staining. The following monoclonal antibodies have been
applied: CD1a (Ortho Diagnostic, Milan, Italy); CD2, CD3, and CD5
(Becton Dickinson, San Jose, CA); CD19, CD20, CD30, and CD45RO (Dako,
Carpinteria, CA); CD25 (IL2R; Boehringer Mannheim, Mannheim,
Germany); CD45RA (Biotest, Milan, Italy); and TCRAB and
TCRGD (T-cell Sciences, Cambridge, UK). Thymic epithelium was
identified with anticytokeratin monoclonal antibody (clone MNF 116; Dako).
Preparation of RNA and cDNA and amplification of TCR segments.
Peripheral blood mononuclear cells were obtained after Ficoll Hypaque
gradient centrifugation. Total cytoplasmic RNA and cDNA for the
analysis of TCRBV and TCRDV chains were prepared from thymus and
peripheral blood mononuclear cells (PBMC) and tissue-infiltrating lymphocytes, as previously described.18 Briefly, 1 to 2 µg of total RNA, prepared by the guanidium
thiocyanate-phenol-chloroform method, were used to synthesize the first
strand of the B and D chain-specific cDNA using a primer specific for
TCRBC1 and TCRBC2 genes ( cDNA: 5' GGG CTG CTC
CTT GAG GGG CTG CGG 3') and a primer specific for the TCRDC
region ( cDNA: 5' CAC TGG GAG AGA GAT GAC AAT AGC AG 3').
TCRBV-specific cDNA was then subjected to enzymatic amplification using
a second TCRBC primer (AI: 5' CCC ACT GTG CAC CTC CTT CC
3') and a TCRBV degenerated primer [Vd: 5' ACG TGA ATT
CT(GT) T(ACT)(CT) TGG TA(CT) (AC) (AG)(AT) CA 3'] that was designed to amplify B-chain rearrangements containing virtually all
of the known human TCRBV genes.19 After PCR
amplifications, the specificity of the total TCRBV amplified products
was analyzed using a colorimetric method and biotinylated
TCRBV-specific probes.20 Subsequently, the TCRBV chains of
interest were amplified by 35 cycles of PCR, using TCRBV-specific
oligonucleotides (TCRBV1: 5' GCA CAA CAG TTC CCT GAC TTG CAC
3'; TCRBV2: 5' TCA TCA ACC ATG CAA GCC TGA CCT 3';
TCRBV3: 5' GTC TCT AGA GAG AAG AAG GAG CGC 3'; TCRBV4:
5' GCC CAA ACC TAA CAT TCT CAA CTC 3'; TCRBV5S1: 5' ATA CTT CAG TGA GAC ACA GAG AAA 3'; TCRBV5S2: 5'
TTC CCT AAC TAT AGC TCT GAG CTG 3'; TCRBV6: 5' AGG CCT GAG
GGA TCC GTC TC 3'; TCRBV8: 5' ATT TAC TTT AAC AAC AAC GTT
CCG 3'; TCRBV13S1: 5' CAA GGA GAA GTC CCC AAT
3'; TCRBV13S2: 5' GGT GAG GGT ACA ACT GCC 3';
TCRBV14: 5' GTC TCT CGA AAA GAG AAG AGG AAT 3'; TCRBV15: 5' AGT GTC TCT CGA CAG GCA CAG GCT 3'; TCRBV17: 5'
AGA TAT AGC TGA AGG GTA CAG CGT 3'; TCRBV20: 5' AGC TCT GAG
GTG CCC CAG AAT CTC 3') and the TCRBC primer (AI).
Aliquots of the TCRDV reverse transcription reaction were amplified
with primers specific for TCRDV1 (official designation TCRDV101S1:
5' TCG CCA GGG TTC TGA TGA ACA GAA 3'), TCRDV2 (official designation TCRDV102S1A1T: 5' AGG AAG ACC CAA GGT AAC ACA A
3'), TCRDV3 (official designation TCRDV103S1A1T: 5' GGT ACT
GCT CTG CAC TTA CGA CAC 3'), TCRDV4 (official designation ADV6:
5' AGC CCA GCA GTG GGG AAA TCG A 3'), TCRDV5 (official
designation ADV21: 5' ACC CTG CTG AAG GTC CTA CAC ATT CC
3'), and a second primer recognizing the TCRDC region (c:
5' AAT TCC TTC ACC AGA CAA GCG ACA 3'). After an initial
hot start, amplification consisted of 35 to 40 cycles of 1 minute at
94°C, 1.5 minutes at 58°C, and 1.5 minutes at 72°C for
TCRDV1 and TCRDV3; 1 minute at 94°C, 1.5 minutes at 60°C, and
1.5 minutes at 72°C for TCRVD2 and TCRDV5; and 1 minute at
94°C, 1.5 minutes at 64°C, and 1.5 minutes at 72°C for
TCRVD4, followed by a final extension for 7 minutes at 72°C. The
specificity of these primers was assessed by the use of cDNA from
TCRGD+ leukemia cell lines and lymphocytes of patients with
lymphoproliferative diseases. All primer combinations failed to amplify
a product when cDNA was omitted from the reaction or when irrelevant
cDNA was used as substrate.
Heteroduplex and TCR sequence analysis.
Amplification products obtained from PCR performed with TCRBV- and
TCRDV-specific primers were heated to 95°C for 5 minutes and then
cooled to 50°C for 1 hour. The annealed samples, which were kept on
ice until used, were run for 5 to 6 hours at 200 V at room temperature
on a 12% nondenaturating polyacrylamide gel (PAGE; 29:1
acrylamide/bisacrylamide) performed in 1× TBE buffer (0.089 mol/L
Tris-borate and 0.002 mol/L EDTA, pH 8.0). The gels were stained for 30 to 60 minutes at room temperature in the dark in a solution containing
0.75 µg/mL ethidium bromide in 200 mL of 1× TBE and were then
photographed under UV light. Amplified TCRBV8 products from the T-cell
line J77 and from lymphocytes stimulated with an anti-TCRBV8 segment
monoclonal antibody were used as monoclonal and polyclonal TCRBV
controls.21 TCRBV1-, TCRBV13S1-, TCRBV15-, and
TCRDV3-amplified products from both PBMC and thymus were purified,
cloned, and sequenced as described.22 Sequences were
compared with published data relative to TCRBV, TCRBD, TCRBJ, TCRBC,
TCRDV, TCRDD, TCRDJ, and TCRDC segments.23 D elements were
assigned with the requirement for a minimum of three contiguous matches
to the germline sequence.
| |
RESULTS |
Characterization of the molecular defect.
The analysis of RAG1 and RAG2 genes was performed on
the genomic DNA of the patient and her parents, taking advantage of the absence of introns in the coding region of both genes. Sequence analysis showed homozygosity for a C to T transition at nucleotide 579 of the RAG1 gene, causing a missense mutation (alanine to valine) at codon 156. However, in view of the analysis of this mutant
by Schwarz et al,17 this mutation can be considered as a polymorphism.
The RAG2 gene was amplified in 1 fragment and the product was
directly sequenced. The analysis of the sequence showed the presence on
both alleles of a missense mutation (G to A at the nucleotide 1887),
leading to arginine to glutamine amino acid change at codon 229 (R229Q;
data not shown). To further confirm the presence of this mutation, we
cloned the PCR fragment into the TA vector and sequenced 10 independent
clones. All of the examined clones bore the altered nucleotide. Both of
these mutations were traced back to the phenotypically normal parents,
who were heterozygous for both the RAG1 polymorphism and the
RAG2 mutation. The latter was not identified in
more than 100 independent alleles from control subjects of the same
ethnic origin. A compound heterozygote patient, bearing a deletion of
both RAG1 and RAG2 genes in one allele and the
RAG2 R229Q mutation on the other allele, has been previously
reported by Schwarz et al.17 The R229Q mutant had a
severely decreased, albeit not abolished, V(D)J recombination activity17 and is therefore likely to be responsible for
the Omenn phenotype in our patient.
Histopathology.
The histopathological changes of the lymph node and the skin were
similar to those previously described in OS.12 Briefly, the
lymph node parenchyma showed a moderate depletion of lymphocytes that
were represented exclusively by T cells and were associated with
numerous eosinophils and CD1a+ interdigitating cells. The T
lymphocytes showed a mature peripheral phenotype (CD2+,
CD3+, CD5+), with predominance of
CD45R0+ over naive CD45RA+ cells; scattered
blasts expressed CD25 and CD30. The analysis of the TCR expression
showed that approximately half of the T cells were TCRAB+
and the remaining were TCRGD+.
The spleen, the skin, and the mucosa-associated lymphoid tissues
contained only a few scattered CD3+ T lymphocytes; also, B
cells were totally absent from these tissues.
The thymic parenchyma showed lack of cortico-medullary demarcation and
was largely represented by epithelial cells (stained with
anticytokeratin antibody; Fig 1A), with
depletion of CD3+ T cells (Fig 1B). The number of
CD45R0+ lymphocytes largely exceeded that of
CD45RA+ cells (Fig 1C and D); furthermore, most of T cells
expressed the TCRGD (Fig 1E). The intrathymic nature of T lymphocytes
was proven by the expression of the CD1a antigen (Fig 1F).
View larger version (187K):
[in this window]
[in a new window]
| Fig 1.
Subserial sections of the thymus stained with antibodies
against cytokeratin (A), CD3 (B), CD45R0 (C), CD45RA (D), TCRGD (E),
and CD1a (F). In a thymic lobule, which is largely composed of
epithelial cells, the CD3+ lymphocytes predominantly
express CD45R0 and TCRGD. Many lymphoid cells show delicate membrane
reactivity for CD1a (streptavidin-biotin complex immunoperoxidase
technique, counterstained with Mayer's hematoxylin; original
magnifications: [A] through [E], ×160; [F], ×400).
|
|
Analysis of TCRBV and TCRDV repertoires in PBMC and thymus.
The general pattern of TCRBV usage by PBMC obtained from the patient
did not significantly differ from that of unrelated age-matched healthy
infants of the same ethnic origin16 and from that of the
healthy infant used as a control in this study
(Fig 2A). However, heteroduplex analysis of
highly expressed TCRBV chains showed a truly polyclonal repertoire in
the healthy control infant, but not in the patient. In fact, as shown
in Fig 2B, all TCRBV products obtained from the healthy control's PBMC
migrated as smears in the polyacrylamide gel, indicating the presence
of heterogeneous molecular species, whereas those from OS patients,
with the only exception of TCRBV13S2 segment, resulted in single or
double homoduplex bands in the context of a background of heteroduplex
bands, thus suggesting the existence of mono/oligoclonal T-cell subsets
within all TCRBV segments analyzed. This result is consistent with
previous observations suggesting that the TCR repertoire is highly
restricted in OS and that, at variance with what was found in elderly
individuals,24,25 each TCRBV cell population is fully
polyclonal in healthy infants.
View larger version (62K):
[in this window]
[in a new window]
| Fig 2.
(A) Expression of TCRBV segments by OS ( )
and a healthy age-matched infant ( ) PBMC. The data are expressed as
the percentage of the color- imetric signal obtained with the
individual TCRBV-specific probes. (B) Heteroduplex analysis of the
indicated TCRBV chains' PCR products of PBMC and thymocytes from the
OS patient and the healthy control. On the right of each gel,
monoclonal and polyclonal controls, prepared by loading in the gel the
products obtained from TCRBV8-specific PCR amplification of the RNA of
monoclonal (J77) and polyclonal (C1.632) cell
populations,21 are shown.
|
|
Similarly, whereas in the control thymus most of the TCRBV chains were
polyclonal, a restricted pattern of homo/heteroduplex bands was
observed (although not as clearly as in PBMC) in thymocytes prepared
from the patient.
Using PCRs performed with TCRDV-specific primers followed by the
heteroduplex analysis, a limited diversity was also demonstrated within
TCRDV gene families from the patient's PBMC and thymocytes (Fig 3, left). However, there were some
differences between the 2 compartments: for instance, 2 homoduplex and
several heteroduplex bands were obtained by loading in the
polyacrylamide gel the TCRDV3 amplification product from PBMC, whereas
a single band appeared when starting from thymocytes. Furthermore, all
5 TCRDV segments were amplified from PBMC, whereas thymocytes appeared
to lack the message for the TCRDV5 chain. Smears only, indicative of a fully polyclonal TCRDV repertoire, were generated using PBMC and thymocytes from a healthy control, with the only exception being the
TCRDV4 segment from the thymus that induced the formation of a faint
homoduplex band (Fig 3, right).
View larger version (46K):
[in this window]
[in a new window]
| Fig 3.
Heteroduplex analysis of the indicated TCRDV chains' PCR
products of PBMC and thymocytes from the OS patient and an age-matched
control.
|
|
Taken together, these results suggest that both peripheral and thymic
TCRBV and TCRDV repertoires are highly restricted in the OS patient.
This prompted us to examine the TCR repertoire in more detail.
Therefore, to confirm the mono/oligoclonal nature of T-cell populations
from the OS patient and to establish whether the clones observed in the
peripheral blood were representative of those identified in the thymus,
the nucleotide sequences of some TCRBV and TCRDV segments were
determined in both samples. All the sequenced cDNA clones resulted from
in-frame rearrangements and, therefore, were likely to correspond to
productive TCR transcripts.
We chose to sequence the TCRBV13S1 chain because it appeared to be
clonal in both peripheral and thymic lymphocytes and the TCRBV15
segment because it resulted in 2 very distinct homoduplex bands in the
periphery and in a single band in the thymocytes. We also sequenced the
TCRBV1 PCR product that was detectable only in the periphery and not in
the thymus to ensure that it was not the result of a technical artifact.
The global analysis of the sequences derived from patients' PBMC and
thymocytes showed a predominance of 2 or a few clones within each of
the TCRBV transcripts analyzed (Fig 4). In
particular, the TCRBV13S1 and TCRBV15 clones, both bearing TCRBJ1S5
region, that were predominant in peripheral blood were also found in
the thymus, where, however, they did not represent the prevalent
clones. On the other hand, the TCRBV13S1 clones that were predominantly expressed in the thymus were not represented in the periphery. As
expected, cDNA TCRBV clones generated from thymocytes from a control
infant were free of predominant sequences.
View larger version (53K):
[in this window]
[in a new window]
| Fig 4.
Junctional nucleotide and amino acid TCRBV1, TCRBV13S1,
and TCRBV15 sequences obtained from PBMC and thymocytes from the OS
patient and from the age-matched control (C). Amino acid sequences are
shown on the right side and displayed using the standard 1-letter code.
Only the last amino acids of the TCRBV chains and the first 5'
amino acids of TCRBJ segments are shown. N/P, nontemplated and
palindromic nucleotides; freq, number of each clone in the total cDNA
clones characterized; °, junctions in which D regions cannot be
assigned; *, clone with the following N nucleotides:
CTGTCTCCCCGCATGTTAGCTCCTC.
|
|
Sequence analysis of TCRDV chains demonstrated that the degree of
diversity of the TCRDV repertoire in the OS patient appeared to be
slightly different from that of TCRBV. Indeed, despite the presence of
dominant clones, a residual polyclonal background could be clearly
documented in the thymus; moreover, in peripheral blood, of the 10 clones sequenced, 3 different sequences were identified
(Fig 5). However, as in the case of TCRBV
sequences, 2 of the clones found in peripheral blood were also detected
in the thymus, whereas the dominant sequences observed in thymocytes were not detectable in the periphery.
View larger version (26K):
[in this window]
[in a new window]
| Fig 5.
Junctional nucleotide and amino acid TCRDV3 sequences
obtained from PBMC and thymocytes from the OS patient and the control
(C). Germline sequences are indicated at the top and are double
underlined. Amino acid sequences are shown on the right side and
displayed using the standard 1-letter code. Only the last amino acids
of the TCRDV chains and the first 5' amino acids of TCRBJ
segments are shown. N/P, nontemplated and palindrome nucleotides; freq,
number of each clone in the total cDNA clones characterized.
|
|
Finally, all of the TCRDV3 sequences from the OS patient were joined in
frame with the TCRDJ1S1 region, but this feature appeared to be a
characteristic of this V segment, because it can be observed also in
all the sequences obtained from the control thymocytes and in other
published sequences.26
TCRBV and TCRDV repertoire diversity of tissue-infiltrating
lymphocytes.
We next investigated the TCRBV and TCRDV repertoires in several other
tissues (skin, lymph node, spleen, bone marrow, and intestine) to
determine whether an oligoclonal pattern was also detected in these
tissues and, if so, to assess whether it recapitulates the pattern
observed in peripheral blood or in the thymus (indicating nonspecific
lymphocytic infiltrates) or whether further compartmentalization of TCR
specificities occurs in different organs and tissues.
Only oligoclonal TCRBV6 and TCRBV13S1 segments were detected in the
colon (data not shown); the other TCRBV-specific primers, which
reproducibly amplified other samples, did not show the presence of
additional TCRBV families in the colon and in the small intestine. All
of the 12 TCRBV families studied could be amplified from the spleen and
bone marrow, and 9 were expressed in the skin. Only 6 transcripts were
detected in the lymph node. The TCRBV13S2 amplicon that was
undetectable in the other samples prepared from the OS patient,
including PBMC and thymus, was identified in the splenic tissue and in the bone marrow. The heteroduplex analysis demonstrated variable patterns of homoduplex/heteroduplex TCRBV bands in the different tissues (Fig 6A), with TCRBV1,
TCRBV2, TCRBV3, TCRBV4, TCRBV5S1, and TCRBV5S2 segments being more
heterogeneous than the other TCRBVs. With regard to TCRGD lymphocytes,
TCRDV1, TCRDV2, and TCRDV3 chain amplified products were obtained from
all tissues analyzed, whereas TCRDV4 and TCRDV5 segments were detected
only in the spleen. Although with different patterns, most of these transcripts migrated in the gel of polyacrylamide as discrete bands of
homo/heteroduplex, thus confirming the restriction of the TCRDV
repertoire also in target organs (Fig 6B). When run in parallel, the
TCRDV1 and TCRDV2 amplification products, which were obtained from
different tissues, showed both common and tissue-specific bands,
indicating that some TCR clonotypes overlap, but that other dominant
clonotypes are preferentially expanded at specific sites (Fig 6C).
View larger version (67K):
[in this window]
[in a new window]
| Fig 6.
Heteroduplex analysis of TCRBV (A) and TCRDV (B) chains
in lymphocytes infiltrating the indicated tissues. In (C), the
amplification products of TCRDV1 and TCRDV2 obtained from different
tissues (T, thymus; S, skin; L, lymph node; Sp, spleen; BM, bone
marrow) are run in parallel.
|
|
Effect of RAG mutations on the V(D)J recombination process in OS.
Among 130 sequences obtained from PBMC and thymocytes from this and 2 other molecularly defined OS patients observed at our institution (OS
patients no. 1 and 4 from Villa et al16), 25 were different
from each other. As compared with a group of 115 sequences prepared
from age-matched children and used as control, these sequences showed a
statistically significant bias (P = .0002) in the TCRBJ2 versus
TCRBJ1 cluster use (Fig 7A, top panel).
Furthermore, when compared with controls, lymphocytes from OS patients
predominantly use the TCRBJ2S7 segment (P = .031); in contrast,
the TCRBJ2S1 and TCRBJ1S1 segments are much less expressed in patients
with OS than in controls (Fig 7A, bottom panel).
View larger version (27K):
[in this window]
[in a new window]
| Fig 7.
(A) (Top) Frequency of TCRBJ1 and TCRBJ2 cluster usage.
TCRJ regions frequencies were calculated from the data set and are
shown here as a histogram. (Bottom) Frequency of individual TCRBJ
usage. (B) Lengths of the CDR3 loops. The number of OS sequences is 25. The number of sequences from age-matched children is 115.
|
|
Because the length and amino acid composition of the antigen-combining
site (CDR3) of the TCR may provide information about the factors
involved in the rearrangement process or about the nature of ligand(s)
that select previously rearranged segments, we compared the length and
amino acid composition of the CDR3 elements of the TCR in the 3 OS
patients reported above and in sequences from age-matched controls. In
both groups, most of CDR3 elements were 9 or 10 amino acids long (Fig
7B), with a broader length range in the control than in the OS group (6 to 18 v 7 to 14). Furthermore, the mean CDR3 length was shorter
in the OS group than in controls (Table
1a); this difference became significant only when the analysis of OS
patients was extended to include all the TCR sequences obtained from OS
patients published so far (this work and previous
studies13-16). However, detailed analysis of the molecular
mechanisms that may modify the structure of the TCR transcripts at the
V-D and D-J junctions failed to show significant abnormalities in
patients with OS versus controls (Table 1c through f), with the
possible exception of a higher frequency of complete V and J elements
(Table 1b). Although common amino acid stretches were not identified
within the CDR3 of individual patients or among different patients,
there was a biased usage of particular amino acids in certain positions
of the CDR3 region (Table 2). As already
reported for murine and human adults' TCR sequences,27,28 glycine (G) is by far the most frequently used residue in
the TCR CDR3 sequences from both OS patients and control children. Moreover, the amino acids serine (S), tyrosine (Y), arginine (R), and
asparagine (N) were significantly more represented at specific positions of the CDR3 in OS patients as compared with sequences from
age-matched controls.
| |
DISCUSSION |
OS is a rare, severe immune deficiency with well-defined clinical and
immunological features that is most often due to mutations in the
RAG genes. In our series, among 8 patients in which a diagnosis of OS was based on typical clinical and immunological features, 6 showed mutations in either RAG1 or RAG2 (Villa et
al16 and data not shown). The residual expression and
function of the RAG mutants identified in most OS patients allow a
partial V(D)J recombination activity16 and account for the
peculiar leakiness of OS as compared with its allelic SCID variant with
undetectable T and B lymphocytes (TB SCID) that is due to severe
RAG gene mutations.17 We have reported a patient
with OS who carried a homozygous mutation in the RAG2 gene
leading to the R229Q amino acid substitution. The same patient was also
homozygous for the alanine to valine substitution at codon 156 of the
RAG1 gene. Both changes have been previously reported by
Schwarz et al17; in particular, the A156V change in the
RAG1 gene was shown not to affect the efficiency of the recombination process using an extrachromosomal V(D)J assay and is
therefore considered a polymorphism. In contrast, the R229Q substitution was reported in 1 allele of the RAG2 gene from an SCID patient who carried a complete deletion encompassing both RAG1 and RAG2 genes on the second allele. When
transfected into a human fibroblast cell line, the RAG2 R229Q
mutant was found to induce RAG2 protein expression at comparable levels
as the wild-type RAG2 construct; however, when assayed for in
vitro V(D)J recombination, the R229Q mutant showed profound defects in
both coding and signal joint formation.17 Interestingly,
the immunological phenotype of the patient with the R229Q RAG2
mutation reported by Schwarz et al17 differed from
typical TB SCID because of a
large proportion (59%) of autologous CD3+ lymphocytes. The
possibility of gene reversion was ruled out by mutation analysis.
Moreover, the T lymphocytes from this patient were found to express an
oligoclonal TCRAV and TCRBV repertoire.17 As a whole, our
data and those reported by Schwarz et al17 indicate that
the R229Q RAG2 mutant allows residual V(D)J recombination activity.
Although no data are available on the clinical features in the patient
described by Schwarz et al,17 our patient had typical
clinical and immunological features of OS. Whether the same leaky
mutation in RAG genes may result in a different clinical and
immunological phenotype (depending on background genetic or environmental factors) awaits the investigation of a larger series of patients.
It is known for several years that OS is characterized by an intrinsic
defect of the lymphocyte lineage. In fact, in OS patients, B
lymphocytes are usually absent and the T-cell phenotype and repertoire
are profoundly abnormal. In most cases, T lymphocytes from OS patients
belong to the TCRAB lineage and show an unbalanced CD4/CD8 ratio that
may, however, vary from patient to patient.2,6,7 In a
minority of OS patients, an increased or even predominant proportion of
T cells that express the TCRGD is observed.6,7 Occasionally, OS patients present a predominant TCRAB+
CD4CD8 population.29
Therefore, it appears that CD4/CD8 homeostasis that is under genetic
control in healthy individuals30 may be lost in OS patients.
There are several pieces of evidence suggesting that, independently
from the type of TCR expressed, the T-cell repertoire of OS patients is
highly restricted.6,13-16 This peculiarity has been
recently ascribed to mutations that impair, but do not completely
abolish, the function of RAG1 and RAG2
genes.16 Apparently, all of the TCRBV gene elements could
be used by OS patients to create functional TCR, but the diversity
within each TCRBV family is very limited. The resulting TCR repertoire
strongly differs from the fully polyclonal pattern of healthy children,
but rather resembles that of patients with acute (Epstein-Barr virus)
or chronic (human immunodeficiency virus)
infections.31-34 On the other hand, the possibility that
restriction of the T-cell repertoire in OS is mainly due to chronic
infections occurring in infants with combined immune deficiency is
unlikely, in view of the fact that a polyclonal T-cell repertoire was
identified in patients with major histocompatibility
complex class II deficiency,35 even if
chronically infected (our own data; data not shown).
The demonstration of restricted TCRAB and TCRGD repertoires in the
thymus from the OS patient might either reflect an intrinsic developmental problem within this organ or recirculation and
immigration of a few clonotypes that have undergone peripheral
expansion. To discriminate between the 2 possibilities, we have
compared the T-cell repertoire in the thymus with that from peripheral blood and tissue-infiltrating T lymphocytes. We found that only a few
TCRBV and TCRDV specificities were expressed in thymocytes as well as
in circulating and in tissue-infiltrating lymphocytes. In particular,
the observation that the sequence of the predominantly expressed
specificities are different in the thymus versus the periphery and that
some specificities are uniquely found in the thymus argues against the
possibility that the repertoire identified in the thymus simply
reflects that of circulating T lymphocytes that have re-entered the
thymus. To further prove that the restriction of the T-cell repertoire
identified in the thymus reflects an intrinsic developmental problem
within this organ, rather than recirculation and immigration from the
periphery, we have performed a detailed immunohistochemical analysis to
identify the origin of thymic T cells. These lymphocytes were
predominantly TCRGD+ and CD45R0+; coexpression
of the CD1a antigen indicated their thymic-cortical origin. It should
be noted that, even in normal thymuses, cortical thymocytes are
CD45R0+.36
As a whole, our data indicate that the defect in the RAG2 gene
in this patient resulted in a restricted T-cell repertoire already in
the thymus, but our data do not allow us to distinguish between the
possibility of a primary repertoire restriction event versus selective
activation and expansion (possibly in response to autoantigens) of
individual clonotypes within this organ. The latter hypothesis is
supported by the demonstration that in our patient both thymocytes and
peripheral blood T cells express a similar proportion of different
V-gene families, suggesting that patients with OS have the potentiality
to generate multiple TCR specificities. The residual background
demonstrated in the heteroduplex analysis, particularly within thymic
TCRDV segments, and the occurrence of clonotypes that, although
predominantly expressed in the thymus, are not detectable in the
periphery further indicate active, ongoing intrathymic lymphopoiesis in OS.
We also found that the diversity of some TCR segments appeared to be
different in the various peripheral tissues, with some TCR families,
such TCRBV1 and TCRBV13S2 or TCRDV4 and TCRDV5, being detectable in
some samples, but not in others. Furthermore, comparison of TCRDV1 and
TCRDV2 amplification products obtained from different tissues, showed
that, whereas some TCR clonotypes are widely distributed, others appear
to be tissue-specific. These features argue against a simple
compartmentalization of T-cell clones that are preferentially
rearranged and/or expanded in the thymus, but rather suggest that the
poorly functional and diverse T cells of OS patients may nonetheless be
capable of undergoing antigen-mediated expansion in peripheral tissues.
The higher representation of serine, tyrosine, arginine, and asparagine
at specific CDR3 positions of the 25 distinct clones derived from this
patient and from 2 previously reported patients (patients no. 1 and 4 from Villa et al16) further suggests the possibility of
antigen-driven selection of T lymphocytes in OS. In view of the
clinical features of OS that to some extent resemble what has been
observed in graft-versus-host disease, it has been suggested that the
process of peripheral T-cell expansion is driven by
autoantigens,13 although this has not been formally proven.
The process of V(D)J recombination involves several proteins in
addition to RAGs. In particular, the addition of exogenous nucleotides
(N diversity) mediated by the terminal deoxynucleotidyl transferase
(TdT) or of palindromic sequences (P nucleotides) due to asymmetrical
opening of the hairpin or deletional events results in modification of
the sequence at the border between V-D and D-J segments, thus
contributing to variability of the CDR3 element that plays an important
role in antigen-binding specificity.37,38 The possibility
that RAG genes mutations affect the structure of CDR3 has been
implied by previous observations and assessed in this study. Harville
et al14 found a lack of N nucleotides in TCR sequences from
OS patients, but this finding has not been confirmed in other recent
studies.13,15 However, the OS patients reported in these
studies were not characterized for RAG genes defects, making it
difficult to draw conclusions on the possible effects of RAG
genes on the efficiency of N nucleotides insertions and CDR3
modification. In the series of 25 different TCR sequences from our 3 patients with OS due to RAG defects, we have conclusively shown that
the addition of N nucleotide is intact in OS. A global evaluation of
the structure and length of the CDR3 showed a shorter mean length in
infants with OS as compared with age-matched controls; however, this
difference was statistically significant only when the sequences from
other OS patients (reported by other groups13-15) were
included. Apart from the limit of including patients with unknown
genetic defects, it appears that the difference of the mean CDR3 length
between OS patients and controls is marginal (9.42 v 10.16),
with obvious overlap in the range between the 2 groups. Its real
relevance in antigen-binding may therefore be questioned. Moreover, we
did not find evidence for abnormalities in P nucleotide insertion or in
trimming of the coding elements. Similar observations were made by
Schwarz et al17 in patients with
TB SCID due to RAG
mutations that severely affect RAG protein expression and/or function.
Although the number of coding joints formed by the RAG mutants in an
extrachromosomal V(D)J assay was dramatically reduced as compared with
wild-type RAG proteins, the quality of the rare coding joints,
including nucleotide loss and addition, was indistinguishable from
normals.17 Similarly, although B SCID
patients exhibit an irregular recombination pattern at the JH locus, it has been shown that their recombination
machinery is competent for the addition of P and N
nucleotides.39
The preferential usage of the TCRBJ2 cluster in 25 distinct sequences
from 3 patients with OS (ie, the patient described here and patients
no. 1 and 4 from Villa et al16), rather than indicating preferential association of the mutant RAG proteins with recombination signal sequences (RSS) in this region, is due to the fact that rearrangements occur only through a mechanism that involves deletion of
the intervening sequences; therefore, the TCRBD2S1 segment can only
join to TCRBJ2, whereas TCRBD1S1 may recombine with either the TCRBJ1
or the TCRBJ2 clusters.
In conclusion, we have shown that the generation of an altered T-cell
repertoire in OS reflects both an intrathymic restriction and
peripheral, (auto)antigen-driven expansion. Moreover, the RAG
genes defect that accounts for the disease directly affects early
stages of V(D)J recombination, but does not alter the process of
double-strand-break DNA repair, including N and P nucleotide insertion.
| |
ACKNOWLEDGMENT |
The authors thank Olga Alebardi for her technical assistance.
| |
FOOTNOTES |
Submitted March 23, 1999; accepted July 11, 1999.
Supported by Istituto Superiore di Sanità Grant No. 30A.0.26 (to
L.I.), Telethon Grants No. E.668 (to L.D.N.) and E.495 (to A.V.),
Biomed2 concerted action CT 983007, MURST (co-finanziamento 1997 to
L.D.N.), CNR P.F. Biotecnologie, and Cariplo (paper no. 29).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Luigi D. Notarangelo, MD, Department of
Pediatrics, University of Brescia, Spedali Civili, 25123 Brescia,
Italy; e-mail: notarang{at}master.cci.unibs.it.
| |
REFERENCES |
1.
Omenn GS:
Familial reticuloendotheliosis with eosinophilia.
N Engl J Med
273:427, 1965
2.
Le Deist F, Fischer A, Durandy A, Arnaud-Battandier F, Nezelof C, Hamet M, De Prost Y, Griscelli C:
Deficit immunitaire mixte et grave avec hypereosinophilie.
Arch Fr Pediatr
42:11, 1985[Medline]
[Order article via Infotrieve]
3.
Ochs HD, Davis SD, Mickelson E, Kerner KG, Wedgwood RJ:
Combined immunodeficiency and reticuloendotheliosis with eosinophilia.
J Pediatr
35:463, 1974
4.
Gomez L, Le Deist F, Blanche S, Cavazzana-Calvo M, Griscelli C, Fischer A:
Treatment of Omenn syndrome by bone marrow transplantation.
J Pediatr
127:76, 1995[Medline]
[Order article via Infotrieve]
5.
Businco L, Di Fazio A, Ziruolo MG, Boner AL, Valletta EA, Ruco LP, Vitolo D, Ensoli B, Paganelli R:
Clinical and immunological findings in four infants with Omenn's syndrome: A form of severe combined immunodeficiency with phenotypically normal T cells, elevated IgE, and eosinophilia.
Clin Immunol Immunopathol
44:123, 1987[Medline]
[Order article via Infotrieve]
6.
De Saint-Basile G, Le Deist F, De Villartay JP, Cerf-Bensussan N, Journet O, Brousse N, Griscelli C, Fischer A:
Restricted heterogeneity of T lymphocytes in combined immunodeficiency with hypereosinophilia (Omenn's syndrome).
J Clin Invest
87:1352, 1991
7.
Brugnoni D, Airò P, Facchetti F, Blanzuoli L, Ugazio AG, Cattaneo R, Notarangelo LD:
In vitro cell death of activated lymphocytes in Omenn's syndrome.
Eur J Immunol
27:2765, 1997[Medline]
[Order article via Infotrieve]
8.
Martin JV, Willoughby PB, Giusti V, Price G, Cerezo L:
The lymph node pathology of Omenn's syndrome.
Am J Surg Pathol
19:1082, 1995[Medline]
[Order article via Infotrieve]
9.
Facchetti F, Blanzuoli L, Ungari M, Alebardi O, Vermi W:
Lymph node pathology in primary combined immunodeficiency diseases.
Springer Semin Immunopathol
19:459, 1998[Medline]
[Order article via Infotrieve]
10.
Melamed I, Cohen A, Roifman CM:
Expansion of CD3CD4CD8 T cell population expressing high levels of IL-5 in Omenn's syndrome.
Clin Exp Immunol
95:14, 1994[Medline]
[Order article via Infotrieve]
11.
Schandené L, Ferster A, Mascart-Lemone F, Crusiaux A, Gérard C, Marchant A, Lybin M, Velu T, Sariban E, Goldman M:
T helper 2-like cells and therapeutic effects of interferon- in combined immunodeficiency with hypereosinophilia (Omenn's syndrome).
Eur J Immunol
23:56, 1993[Medline]
[Order article via Infotrieve]
12.
Chilosi M, Facchetti F, Notarangelo LD, Romagnani S, Del Prete G, Almerigogna F, De Carli F, Pizzolo G:
CD30 cell expression and abnormal soluble CD30 serum accumulation in Omenn's syndrome: Evidence for a T helper-2 mediated condition.
Eur J Immunol
26:329, 1996[Medline]
[Order article via Infotrieve]
13.
Rieux-Laucat F, Bahadoran P, Brousse N, Selz F, Fischer A, Le Deist F, De Villartay JP:
Highly restricted human T cell repertoire in peripheral blood and tissue-infiltrating lymphocytes in Omenn's syndrome.
J Clin Invest
102:312, 1998[Medline]
[Order article via Infotrieve]
14.
Harville TO, Adams DM, Howard TA, Ware RE:
Oligoclonal expansion of CD45R0+ T lymphocytes in Omenn syndrome.
J Clin Immunol
17:322, 1997[Medline]
[Order article via Infotrieve]
15.
Brooks EG, Filipovich AH, Padgett JW, Mamlock R, Goldblum RM:
T-cell receptor analysis in Omenn's syndrome: Evidence for defects in gene rearrangement and assembly.
Blood
93:242, 1999[Abstract/Free Full Text]
16.
Villa A, Santagata S, Bozzi F, Giliani S, Frattini A, Imberti L, Benerini Gatta L, Ochs HD, Schwarz K, Notarangelo LD, Vezzoni P, Spanopoulou E:
Partial V(D)J recombination activity leads to Omenn syndrome.
Cell
93:885, 1998[Medline]
[Order article via Infotrieve]
17.
Schwarz K, Gauss GH, Ludwig LL, Pannicke U, Li Z, Lindner D, Friedrich W, Seger RA, Hansen-Hagge TE, Desiderio S, Lieber MR, Bartram C:
RAG mutations in human B cell-negative SCID.
Science
274:97, 1996[Abstract/Free Full Text]
18.
Sottini A, Quiròs-Roldan E, Notarangelo LD, Malagoli A, Primi D, Imberti L:
Engrafted maternal T cells in SCID patient express TCRBV segments characterized by a restricted V-D-J junctional diversity.
Blood
85:2105, 1995[Abstract/Free Full Text]
19.
Martin R, Howell MD, Jaraquemada D, Flerlage M, Richert J, Brostoff S, Long EO, McFarlin DE, McFarland HF:
A myelin basic protein peptide is recognized by cytotoxic T cells of HLA-DR types associated with multiple sclerosis.
J Exp Med
173:19, 1991[Abstract/Free Full Text]
20.
Bettinardi A, Imberti L, Sottini A, Primi D:
Analysis of amplified T cell receptor V transcripts by a non isotopyc immunoassay.
J Immunol Methods
146:71, 1992[Medline]
[Order article via Infotrieve]
21.
Sottini A, Quiròs-Roldan E, Albertini A, Primi D, Imberti L:
Assessment of T cell receptor variable beta chains diversity by heteroduplex analysis.
Hum Immunol
48:12, 1996[Medline]
[Order article via Infotrieve]
22.
Imberti L, Sottini A, Signorini S, Gorla R, Primi D:
Oligoclonal CD4+CD57+ T-cell expansions contribute to the imbalanced T-cell receptor repertoire of rheumatoid arthritis patients.
Blood
89:2822, 1997[Abstract/Free Full Text]
23.
Arden B, Clark SP, Kabelitz D, Mak TW:
Human T-cell receptor variable gene segment families.
Immunogenetics
42:455, 1995[Medline]
[Order article via Infotrieve]
24.
Hingorani R, Monteiro J, Pergolizzi R, Furie R, Chartash E, Gregersen PK:
CDR3 lenght restriction of T-cell receptor chains in CD8+ T-cells of rheumatoid arthritis patients.
Ann NY Acad Sci
756:179, 1995[Medline]
[Order article via Infotrieve]
25.
Monteiro J, Hingorani R, Choi I-H, Silver J, Pergolizzi R, Gregersen PK:
Oligoclonality in the human CD8+ T cell repertoire in normal subjects and monozygotic twins: Implications for studies of infectious and autoimmune diseases.
Mol Med
1:614, 1995[Medline]
[Order article via Infotrieve]
26.
Holtmeier W, Rowell DL, Nyberg A, Kagnoff MF:
Distinct T cell receptor repertoires in monozygotic twins concordant for coeliac disease.
Clin Exp Immunol
107:148, 1997[Medline]
[Order article via Infotrieve]
27.
Candéias S, Waltzinger C, Benoist C, Mathis D:
The V17+ T cell repertoire: Skewed J usage after thymic selection; dissimilar CDR3s in CD4+ versus CD8+ cells.
J Exp Med
174:989, 1991[Abstract/Free Full Text]
28.
Quiròs Roldan E, Sottini A, Bettinardi A, Albertini A, Imberti L, Primi D:
Different TCRBV genes generate biased patterns of V-D-J diversity in human T cells.
Immunogenetics
41:91, 1995[Medline]
[Order article via Infotrieve]
29.
Wirt DP, Brooks EG, Vaidya S, Klimpel GR, Waldmann TA, Goldblum RM:
Novel T-lymphocyte population in combined immunodeficiency with features of graft versus host disease.
N Engl J Med
321:370, 1989[Medline]
[Order article via Infotrieve]
30.
Amadori A, Zamarchi R, De Silvestro G, Forza G, Cavatton G, Danieli GA, Clementi M, Chieco-Bianchi L:
Genetic control of the CD4/CD8 T-cell ratio in humans.
Nat Med
12:1279, 1995
31.
Callan MFC, Steven N, Krausa P, Wilson JDK, Moss PAH, Gillespie GM, Bell JI, Rickinson AB, McMichael AJ:
Large clonal expansions of CD8+ T cells in acute mononucleosis.
Nat Med
2:906, 1996[Medline]
[Order article via Infotrieve]
32.
Bettinardi A, Imberti L, Sottini A, Quiròs-Roldan E, Puoti M, Castelli F, Cadeo GP, Gorla R, Primi D:
Detection of clonal T cell populations with closely related T cell receptor junctional sequences in persons at high risk for human immunodeficiency virus (HIV) infection and in patients acutely infected with HIV.
J Infect Dis
175:272, 1997[Medline]
[Order article via Infotrieve]
33.
Connors M, Kovacs JA, Krevat S, Gea-Banacloche JC, Sneller MC, Flanigan M, Metcalf JA, Walker RE, Falloon J, Baseler M, Stevens R, Feuerstein I, Masur H, Lane HC:
HIV infection changes in CD4+ T-cell phenotype and depletions within the CD4+ T-cell repertoire that are not immediately restored by antiviral or immune-based therapies.
Nat Med
3:533, 1997[Medline]
[Order article via Infotrieve]
34.
Pantaleo G, Demarest JF, Soudeyns H, Graziosi C, Denis F, Adelsberger JW, Borrow P, Saag MS, Shaw JM, Sekaly RP, Fauci AS:
Major expansion of CD8+ T cells with a predominant V usage during the primary immune response to HIV.
Nature
370:463, 1994[Medline]
[Order article via Infotrieve]
35.
van Eggermond MC, Rijkers GT, Kuis W, Zegers BJ, van den Elsen PJ:
T cell development in a major histocompatibility class II-deficient patient.
Eur J Immunol
23:2585, 1993[Medline]
[Order article via Infotrieve]
36.
Gillitzer R, Pilarski LM:
In situ localization of CD45 isoforms in the human thymus indicates a medullary location for the thymic generative lineage.
J Immunol
144:66, 1990[Abstract]
37.
Jorgensen JL, Esser U, Fazekas de St Groth B, Reay PA, Davis MM:
Mapping T-cell receptor-peptide contacts by variant peptide immunization of single-chain transgenics.
Nature
355:224, 1992[Medline]
[Order article via Infotrieve]
38.
Garboczi DN, Ghosh P, Utz U, Fan QR, Biddison WE, Wiley DC:
Structure of the complex between human T-cell receptor, viral peptide and HLA-A2.
Nature
384:134, 1996[Medline]
[Order article via Infotrieve]
39.
Schwarz K, Hansen-Hagge TE, Knobloch C, Friedrich W, Kleihauer E, Bartram C:
Severe combined immunodeficiency (SCID) in man: B cell-negative (B) SCID patients exhibit an irregular recombination pattern at the JH locus.
J Exp Med
174:1039, 1991[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
P. L. Poliani, F. Facchetti, M. Ravanini, A. R. Gennery, A. Villa, C. M. Roifman, and L. D. Notarangelo
Early defects in human T-cell development severely affect distribution and maturation of thymic stromal cells: possible implications for the pathophysiology of Omenn syndrome
Blood,
July 2, 2009;
114(1):
105 - 108.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S.-Y. Wong, C. P. Lu, and D. B. Roth
A RAG1 Mutation Found in Omenn Syndrome Causes Coding Flank Hypersensitivity: A Novel Mechanism for Antigen Receptor Repertoire Restriction
J. Immunol.,
September 15, 2008;
181(6):
4124 - 4130.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Schuetz, K. Huck, S. Gudowius, M. Megahed, O. Feyen, B. Hubner, D. T. Schneider, B. Manfras, U. Pannicke, R. Willemze, et al.
An Immunodeficiency Disease with RAG Mutations and Granulomas
N. Engl. J. Med.,
May 8, 2008;
358(19):
2030 - 2038.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Matangkasombut, M. Pichavant, D. E. Saez, S. Giliani, E. Mazzolari, A. Finocchi, A. Villa, C. Sobacchi, P. Cortes, D. T. Umetsu, et al.
Lack of iNKT cells in patients with combined immune deficiency due to hypomorphic RAG mutations
Blood,
January 1, 2008;
111(1):
271 - 274.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. V. Gulino, D. Moratto, S. Sozzani, P. Cavadini, K. Otero, L. Tassone, L. Imberti, S. Pirovano, L. D. Notarangelo, R. Soresina, et al.
Altered leukocyte response to CXCL12 in patients with warts hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome
Blood,
July 15, 2004;
104(2):
444 - 452.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E Hodges, M T Krishna, C Pickard, and J L Smith
Diagnostic role of tests for T cell receptor (TCR) genes
J. Clin. Pathol.,
January 1, 2003;
56(1):
1 - 11.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Giovannetti, F. Mazzetta, E. Caprini, A. Aiuti, M. Marziali, M. Pierdominici, A. Cossarizza, L. Chessa, E. Scala, I. Quinti, et al.
Skewed T-cell receptor repertoire, decreased thymic output, and predominance of terminally differentiated T cells in ataxia telangiectasia
Blood,
December 1, 2002;
100(12):
4082 - 4089.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. W. Langerak, R. van den Beemd, I. L. M. Wolvers-Tettero, P. P. C. Boor, E. G. van Lochem, H. Hooijkaas, and J. J. M. van Dongen
Molecular and flow cytometric analysis of the V{beta} repertoire for clonality assessment in mature TCR{alpha}{beta} T-cell proliferations
Blood,
July 1, 2001;
98(1):
165 - 173.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Corneo, D. Moshous, T. Gungor, N. Wulffraat, P. Philippet, F. L. Deist, A. Fischer, and J.-P. de Villartay
Identical mutations in RAG1 or RAG2 genes leading to defective V(D)J recombinase activity can cause either T-B-severe combined immune deficiency or Omenn syndrome
Blood,
May 1, 2001;
97(9):
2772 - 2776.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. J. Sadofsky
The RAG proteins in V(D)J recombination: more than just a nuclease
Nucleic Acids Res.,
April 1, 2001;
29(7):
1399 - 1409.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Villa, C. Sobacchi, L. D. Notarangelo, F. Bozzi, M. Abinun, T. G. Abrahamsen, P. D. Arkwright, M. Baniyash, E. G. Brooks, M. E. Conley, et al.
V(D)J recombination defects in lymphocytes due to RAG mutations: severe immunodeficiency with a spectrum of clinical presentations
Blood,
January 1, 2001;
97(1):
81 - 88.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. A. Gomez, L. M. Ptaszek, A. Villa, F. Bozzi, C. Sobacchi, E. G. Brooks, L. D. Notarangelo, E. Spanopoulou, Z. Q. Pan, P. Vezzoni, et al.
Mutations in Conserved Regions of the Predicted RAG2 Kelch Repeats Block Initiation of V(D)J Recombination and Result in Primary Immunodeficiencies
Mol. Cell. Biol.,
August 1, 2000;
20(15):
5653 - 5664.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
J. G. Noordzij, N. S. Verkaik, N. G. Hartwig, R. de Groot, D. C. van Gent, and J. J. M. van Dongen
N-terminal truncated human RAG1 proteins can direct T-cell receptor but not immunoglobulin gene rearrangements
Blood,
July 1, 2000;
96(1):
203 - 209.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION