Previous Article | Next Article 
Clinical and Diagnostic Laboratory Immunology, November 2000, p. 953-959, Vol. 7, No. 6
1071-412X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
T-Cell Receptor V
Repertoire CDR3 Length Diversity Differs
within CD45RA and CD45RO T-Cell Subsets in Healthy and Human
Immunodeficiency Virus-Infected Children
Zhong Chen
Kou,
Joshua S.
Puhr,
Mabel
Rojas,
Wayne T.
McCormack,
Maureen M.
Goodenow, and
John W.
Sleasman*
Department of Pediatrics, Division of Immunology and Infectious
Diseases, and Department of Pathology, Immunology, and Laboratory
Medicine, University of Florida College of Medicine, Gainesville,
Florida
Received 3 May 2000/Returned for modification 11 July 2000/Accepted 3 August 2000
 |
ABSTRACT |
The T-cell receptor (TCR) CDR3 length heterogeneity is formed
during recombination of individual V
gene families. We
hypothesized that CDR3 length diversity could be used to assess the
fundamental differences within the TCR repertoire of CD45RA and CD45RO
T-cell subpopulations. By using PCR-based spectratyping, nested primers for all 24 human V families were developed to amplify CDR3 lengths in immunomagnetically selected CD45RA and CD45RO subsets within both
CD4+ and CD8+ T-cell populations. Umbilical
cord blood mononuclear cells or peripheral blood mononuclear cells
obtained from healthy newborns, infants, and children, as well as
human immunodeficiency virus (HIV)-infected children, were analyzed.
All T-cell subsets from newborn and healthy children demonstrated a
Gaussian distribution of CDR3 lengths in separated T-cell subsets. In
contrast, HIV-infected children had a high proportion of predominant
CDR3 lengths within both CD45RA and CD45RO T-cell subpopulations, most
commonly in CD8+ CD45RO T cells. Sharp differences in
clonal dominance and size distributions were observed when cells were
separated into CD45RA or CD45RO subpopulations. These differences were
not apparent in unfractionated CD4+ or CD8+ T
cells from HIV-infected subjects. Sequence analysis of predominant CDR3 lengths revealed oligoclonal expansion within individual V
families. Analysis of the CDR3 length diversity within CD45RA and
CD45RO T cells provides a more accurate measure of disturbances in the
TCR repertoire than analysis of unfractionated CD4 and CD8 T cells.
| |
INTRODUCTION |
During T-cell development the
T-cell receptor (TCR) chain is formed by the recombination of
germ line-encoded variable (V)-, diversity (D)-, and joining (J)-region
gene elements (23, 31). The hypervariable region of
each TCR V family, or the complementarity-determining region (CDR3),
is formed by the joining of V-(D)-J segments, thereby establishing the diversity of the cellular immune response (14, 16, 24). Additional diversity is generated within CDR3 through the removal or insertion of non-germ-line nucleotides at each joining
junction (16, 24). Therefore, the TCR CDR3 region varies in
both length and amino acid sequence. This variability can discriminate
between functionally distinct TCR clonotypes. Changes in CDR3 length
and amino acid sequences can be used to monitor the course of the
T-cell response to an antigen (12).
Human CD4+ T cells can be subdivided into subsets of naive
or memory T cells on the basis of the expression of either the high- or
low-molecular-weight isoforms of the leukocyte common antigen, CD45RA
or CD45RO, respectively (8). The majority of T cells in
infants are derived from the thymus and express CD45RA (11), in contrast to adults, in whom the majority of T cells express the
CD45RO T-cell phenotype (29). There is a postthymic
differentiation pathway in which T cells lose the ability to express
CD45RA and gain the ability to express CD45RO after an encounter with
antigen (1, 8, 9). We hypothesized that the distribution of
CDR3 lengths within any particular V family would reflect this
fundamental difference between CD45RA and CD45RO T cells. Using
spectratyping, we developed a highly accurate and sensitive assay to
measure differences in the distribution of CDR3 lengths in highly
purified CD45RA and CD45RO T cells from healthy and human
immunodeficiency virus (HIV)-infected children. Umbilical cord blood
mononuclear cells provided a model for new thymic T-cell emigrants with
a polyclonal TCR diversity (19). HIV-infected subjects
served as a model to assess clonal dominance within T-cell
subpopulations because of the marked skewing of the CDR3 length within
CD4+ and CD8+ T cells from these individuals
(10, 17, 30, 32). Spectratyping in combination with cell
separation techniques that fractionate CD45RA and CD45RO T-cell subsets
distinguish the extent of TCR diversity within individual V families
more accurately and with greater sensitivity than analysis of
unfractionated T cells. Sequence analysis of CDR3 length diversity in
CD45RA and CD45RO T-cell subpopulations can be used to define the
extent of clonal expansion within the TCR repertoire.
| |
MATERIALS AND METHODS |
Subjects.
The study consisted of a cross-sectional analysis
of umbilical cord or peripheral blood samples obtained from three
healthy newborns, two infants, and two children. Additional samples
were obtained from four HIV-infected children at different Centers for
Disease Control and Prevention (CDC) clinical and immune stages of HIV
disease prior to the initiation of antiretroviral therapy. Blood
samples were obtained by a protocol approved by the Institutional Review Board of the University of Florida.
Isolation and purification of T-lymphocyte subsets.
Umbilical cord blood mononuclear cells and peripheral blood mononuclear
cells (PBMCs) were isolated by Ficoll-Hypaque centrifugation. Separation of T-cell subpopulations from umbilical cord blood and
PBMCs was carried out by previously described methods
(2, 28). CD4+ T lymphocytes were selected by
using magnetic multisort microbeads coated with an anti-CD4 monoclonal
antibody (Miltenyi Biotech, Auburn, Calif.) and a magnetic cell-sorting
(MACS) high-gradient magnetic separation column. Microbeads coated with
monoclonal antibody and mononuclear cells were mixed and incubated at
4°C for 15 min. Labeled CD4+ cells which adhered to the
column were washed three times with wash buffer and collected.
CD8+ T cells were selected after depletion of the
CD4+ cells by using microbeads coated with anti-CD8
monoclonal antibody (Miltenyi Biotech) and the MACS magnetic separation
column. Purified CD4+ or CD8+ cells were
released from the beads by using release reagent at 4°C for 15 min.
The T-cell subpopulations were subsequently separated into
CD45RA+ or CD45RO+ subpopulations by using the
microbeads coated with the appropriate monoclonal antibodies (Miltenyi
Biotech). The purity of each T-cell subpopulation was >90%, as
determined by flow cytometry analysis.
Extraction of mRNA and synthesis of cDNA.
Lymphocyte mRNA
was isolated and purified by previously published techniques (6,
7). Briefly, mRNA was extracted from 1 × 105 to
5 × 105 purified T lymphocytes by using guanidinium
isothiocyanate and a spin column with oligo(dT)-cellulose (mRNA
purification kit; Pharmacia, Piscataway, N.J.). First-strand cDNA was
synthesized by mixing mRNA with 1 µg of random hexanucleotides
(Promega, Madison, Wis.) and 5 µl of supertranscriptase II (Gibco
BRL, Grand Island, N.Y.) in a total volume of 20 µl at 37°C for
1 h. The reaction was terminated by heating at 70°C for 10 min.
PCR amplification of CDR3 cDNA.
CDR3 size analysis within
the TCR chain was performed by a two-step PCR as described
previously (5, 21). Separate amplification reactions for
each of 24 human V gene families were carried out in 25-µl
reaction mixtures containing 0.5 µl (20 µM) each of the forward
V primer and the reverse C primer, 0.5 µl (10 mM) of each
deoxynucleoside triphosphate, 2.5 µl of 10× PCR buffer, 20.5 µl of
sterile H2O, 0.25 µl of cDNA, and 1.25 U of
Taq polymerase. Amplifications were started with an initial
denaturation step of 3 min at 95°C, followed by 35 cycles of 95°C
for 1 min, 55°C for 1 min, and 72°C for 1 min, with a final
extension step of 7 min. Primer sequences are shown in Table
1.
The second-round PCR used the first-round products as the template and
a pair of nested PCR primers (Table
1); the forward
primer (V
NS) was
nested 3' to the first-round V
-specific primer,
and the reverse
primer (C
NS) was located 88 bp away from the
CDR3 region. The
reverse primer (C
NS) contained a nontemplate
sequence,
GTTTCTT, on the 5' end, which was labeled at the 5'
end with
a blue fluorescent dye (6-FAM). To increase the amplification
specificity, the primer concentrations were reduced to 5 µM in
a
25-µl reaction mixture. Second-round amplification was performed
at
94°C for 30 s, 55°C for 30 s, and 72°C for 30 s
for 20
cycles.
Analysis of CDR3 length by spectratyping.
Fluorescent PCR
products and a size marker (ROX400; Perkin-Elmer Biosystems, Foster
City, Calif.) were mixed with formamide and were denatured at 94°C
for 2 mins. Samples were loaded onto a 6% acrylamide sequencing gel
(National Diagnostic, Atlanta, Ga.) on a 24-lane Applied Biosystems
model 373 DNA sequencer and were run for 6 h. The data were
analyzed and quantified by using ABIPRISMGeneScan analysis software,
which calculates the relative intensity of each product to generate a
curve for each V family.
Molecular cloning and sequencing of CDR3 segments.
First-round PCR products were amplified with identical forward and
reverse V PCR primers but without the fluorescent label used for
spectratyping of individual families. The second-round products were
used for TA cloning and sequencing. The PCR products were purified by
using a gel extraction kit (QIAGEN, Valencia, Calif.) according to the
manufacturer's recommendation. They were directly cloned into TA
cloning vector PCR 2.1 (Invitrogen Corporation, Carlsbad, Calif.),
sequenced with fluorescent dideoxy terminators, and analyzed on an
Applied Biosystems model 337A automated sequencer. The CDR3 amino acid
sequence was analyzed by using DNAMAN software.
| |
RESULTS |
PCR-based spectratyping of CDR3 length diversity in T-cell lines,
PBMCs, and umbilical cord mononuclear cells.
Nested
forward primers for each of the 24 human V gene families were
designed and used in combination with a nested (CNS) reverse primer
for PCR amplification of the TCR CDR3 region. The amplified CDR3 length
was defined as the region between the upstream amino acid CASS residues
in the V segments and the downstream GXG box in the J region
(31). Jurkat cells, T cells that express TCR V8 and
that have a CDR3 length of 11 amino acids, were used as a control
to optimize the spectratyping analysis (27).
Amplification of Jurkat cDNA with V8 and C primers produced a
single peak. Sequence analysis of the cloned Jurkat PCR products
revealed that 10 of 10 clones had the identical sequence (CASSFSTCSANYGYTFGSG).
Amplification of cDNA from umbilical cord blood samples or
PBMCs from healthy children resulted in CDR3 patterns that
consisted
of 7 to 10 different peaks for each V
family (Fig.
1A and
B).
Each peak in the CDR3 size patterns
was spaced by three nucleotides
(e.g., 1 amino acid). As shown in Fig.
1, all 24 V
families could
be amplified with these primers. Repeated
spectratyping analysis
of the same cDNA samples revealed the identical
patterns of the
TCR V
CDR3 sizes (data not shown).
View larger version (47K):
[in this window]
[in a new window]
|
FIG. 1.
Spectratyping of CDR3 sizes for all 24 V gene
families in healthy newborns and children. Two representative CDR3 size
analyses for the 24 human V gene families are shown for cDNAs
obtained from umbilical cord T cells from a healthy newborn infant (A)
and cDNAs obtained from T cells obtained from a healthy 12 year old
(B). CDR3 size is shown on the x axis, and relative peak
intensity (fluorescence intensity [FI]) is shown on the y
axis. The peak size corresponding to CDR3 lengths of 10 amino acids are
shown for each V family.
|
|
Quantitation of amplified CDR3 lengths within individual V
families.
The intensity of an individual peak reflects the
relative representation of the particular CDR3 sizes within the
individual V family. The relative fluorescence intensity for
individual CDR3 sizes can be quantified as the area under each peak
(17). The data, when expressed as a percentage of the total
area under the curves for each V family, can be used to accurately
determine the degree of CDR3 length diversity within any particular
V family. This analysis of length differences allows comparisons of
TCR diversity among different V families or among multiple
individuals. A representative analysis of the size distribution of CDR3
lengths from CD8+ CD45RO T cells obtained from a healthy
child reveals a Gaussian-like distribution that ranges from 7 to 15 amino acids in length (Fig. 2A). Extremes
in CDR3 lengths make up a minority (<1%) of the size diversity, while
CDR3 sizes of 10, 11, and 12 amino acids make up 18, 24, and 23% of
the area under the curve, respectively. In contrast, an HIV-infected
subject, whose CDR3 size distribution demonstrates clonal dominance,
has a predominant peak of eight amino acids in length, which
encompasses over 80% of the area under the curve (Fig. 2A). All four
HIV-infected children displayed perturbations within their
CD8+ CD45RO T-cell subpopulations. The number of Vß
families exhibiting oligoclonal expansion were 50, 29, 58, and 75% in
the four children, respectively (data not shown). In contrast, in
healthy children less than 5% of the Vß families from
CD8+ CD45RO T cells showed perturbations.
View larger version (17K):
[in this window]
[in a new window]
|
FIG. 2.
Quantitation and sequence analysis of populations of
CDR3 size fragments in healthy and HIV-infected individuals. (A)
Relative distribution of CDR3 size distribution in CD8+
CD45RO T cells from two representative subjects. The upper panel is
CDR3 sizes in V16 from a healthy child, and the lower panel is CDR3
sizes in V16 from T cells from an HIV-infected child. The relative
contribution of each CDR3 size (in amino acid [AA] length) is
determined by the relationship of the peak to the total area under the
curve. The results are shown as a percentage, as determined with
ABIPRISM Gene Scan software. (B) Frequency of individual CDR3
clonotypes within CD8+ CD45RO T cells from the HIV-infected
subject whose results are shown in the lower panel of panel A. In a
separate PCR amplification, the products for V16 were cloned and 12 individual clones were sequenced. Nine of the 12 clones (75%) were 9 amino acids in length and had identical N1-D-N2 and J amino acid
sequences. The other three clones had different lengths and amino acid
sequences.
|
|
The degree of CDR3 size variability in umbilical cord mononuclear cells
was determined to test the reproducibility of the
analysis when applied
to a group of healthy newborn subjects,
who should have similar CDR3
size distributions and for whom there
should be minimal variation
between families or between subjects.
The mean percentage and standard
deviations for each CDR3 size
among the three infants are summarized
for seven representative
V
families (Table
2). The CDR3 size distributions for all
24
V
families showed similar Gaussian distributions.
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Mean percentage and standard deviations for each CDR3
size among three healthy infants in seven representative V families
|
|
Sequence analysis of predominant CDR3 lengths.
A predominant
peak in any given CDR3 pattern may reflect oligoclonal expansion of T
cells expressing a particular TCR or a polyclonal collection of T cells
containing multiple TCRs of the same CDR3 size. We determined if
predominant peaks were polyclonal or oligoclonal by directly
sequencing the amplified CDR3 regions. The predominant CDR3 peak
shown in Fig. 2A for the HIV-infected child had a length of 8 amino
acids and accounted for 82% of V16 CDR3 lengths. In separate PCRs
second-round products were sequenced and compared to the sequences of
products from a healthy subject whose CDR3 lengths demonstrated a
Gaussian distribution. Twelve clones from the HIV-infected child were
sequenced and four different amino acid sequences were identified. As
shown in Fig. 2B, 9 of the 12 clones that were 8 amino acids in length
had identical sequences. The sequences obtained from the healthy child
showed no predominant clone (data not shown). Sequence analysis
confirms that the predominant CDR3 peaks represent an oligoclonal
expansion of T cells.
Comparison of CDR3 size distribution on CD45RA and CD45RO T-cell
subpopulations.
The CDR3 length within the 24 V gene
families was determined by two-step PCR amplification of cDNA obtained
from purified CD45RA or CD45RO from both CD4+ and
CD8+ T cells isolated from PBMCs or umbilical
cord blood samples from neonates, healthy children, and HIV-infected
children. Neonatal cord blood samples demonstrated a Gaussian pattern
similar to the results shown in Table 2 within CD4+ CD45RA,
CD4+ CD45RO, CD8+ CD45RA, and
CD8+ CD45RO in all families (data not shown). In
contrast, there were differences in CDR3 size distributions when cDNAs
from CD45RA and CD45RO T cells from healthy and HIV-infected subjects
were compared (Fig. 3). For healthy
children the lengths of CDR3 from both CD4+ and
CD8+ T cells maintained a Gaussian distribution.
View larger version (34K):
[in this window]
[in a new window]
|
FIG. 3.
Differences in CDR3 spectratyping of T-cell
subpopulations for two V families from healthy and HIV-infected
children. T cells from a healthy 5-year-old subject and an age-matched
HIV-infected subject whose HIV infection is CDC stage A1 were separated
into CD4+ CD45RA, CD4+ CD45RO, CD8+
CD45RA, and CD8+ CD45RO T cells. CDR3 sizes are shown on
the x axis, and fluorescence intensity (FI) is shown on the
y axis. The CDR3 amino acid lengths corresponding to 10 amino acids are shown for each V family.
|
|
For all four asymptomatic HIV-infected children we observed predominant
peaks within three or more V
families. In most cases
expanded CDR3
lengths made up more than 50% of the total area
under the curve for
CDR3 sizes within the individual V
family.
For the representative
infected subject whose results are presented
in Fig.
3, the V
2 CDR3
length containing 12 amino acids was the
predominant peak within
CD8
+ CD45RO T cells. Predominant peaks and perturbations
away from
the normal Gaussian distribution were also apparent within
CD4
+ CD45RO T cells and CD8
+ CD45RO T cells for
V
11 CDR3 lengths for this same subject. This
degree of perturbation
was undetectable in T-cell subpopulations
from the age-matched healthy
child.
Optimization of our ability to detect CDR3 size perturbations
within individual V
families required further separation
of
CD4
+ and CD8
+ T cells into CD45RA and CD45RO
subpopulations. As demonstrated
in Fig.
4, clonal expansions within
CD4
+ or CD8
+ T cells were not evident unless
the cells were further fractionated.
For the HIV-infected subject whose
results are shown in Fig.
4,
CD4
+ and CD8
+ T
cells expressing V
16 and V
7, respectively, did not appear
to contain perturbations unless they were fractionated into CD45RA
and CD45RO subpopulations. Separation clearly shows the
predominant
peak within the CD45RO subsets of both the CD4
+
and CD8
+ T-cell populations.
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 4.
Separation of CD8+ T cells and
CD4+ T cells into CD45RA and CD45RO subpopulations
distinguishes differences in CDR3 length distributions.
CD4+ and CD8+ T cells from an HIV-infected
child were further separated into CD45RA and CD45RO T cells, and the
results were compared to the spectratyping analysis of CDR3 lengths
from unseparated T cells. CDR3 sizes are shown on the x
axis, and fluorescence intensity (FI) is shown on the y
axis. The CDR3 amino acid lengths corresponding to 10 amino acids are
shown for each V family.
|
|
| |
DISCUSSION |
Analysis of TCR CDR3 length by PCR-based spectratyping provides a
powerful tool for the assessment of human TCR diversity in vivo and the
evaluation of TCR diversity in health and disease states (4, 13,
18, 22, 25). In previous studies analysis of CDR3 size diversity
has been focused on evaluations of unfractionated CD4+ and
CD8+ T cells (6, 15, 17, 25, 30). In addition,
the availability of primers for all 24 human V families has been
limited (13, 20). Many V forward primers have failed to
provide the degree of resolution needed to allow distinct
definition and quantitation of individual CDR3 lengths. These
limitations have led to the underutilization of TCR spectratyping in
assessments of T-cell disorders. We have benefited from these
earlier studies and have developed PCR primers and amplification
conditions that result in accurate and reproducible CDR3
spectratyping of the 24 human V families. Our modifications allow
assessment of the relative diversity of CDR3 lengths within individual
V families and comparison of results between healthy and immune
compromised individuals.
Our success in obtaining a high resolution between CDR3 peaks is due to
the specificity of the V forward primers and the placement of the
seven nontemplate nucleotides on the 5' ends of the reverse primers
(3'-CNS). These additional nucleotides ensure the catalytic addition
of nontemplate nucleotides to the 3' ends of the amplified products by
Taq DNA polymerase. The 7-bp nucleotides also result in a
high level of adenylation of the 3' end of the forward DNA strand to
facilitate accurate spectratyping and TA cloning (3). We
were able to confirm both the accuracy and the specificity of the
primers and conditions in preliminary studies using Jurkat cell cDNA as
a model for monoclonal T cells. Umbilical cord blood mononuclear cells
served as a model for polyclonal T cells. Analysis of cDNA obtained
from cord blood mononuclear cells and lymphocytes from healthy children
showed that our spectratyping conditions are highly accurate and
reproducible. The results showed very little skewing of CDR3 length
distributions and only a small degree of variation between
healthy subjects. The V family CDR3 length distribution
commonly conformed to a Gaussian distribution. The distributions of the
percentages of relative peaks among the different CDR3 sizes in
umbilical cord blood mononuclear cells and T cells from healthy
children were remarkably similar.
The methods used to separate T-cell subpopulations were effective in
achieving a significant degree of cell purity, allowing us to evaluate
CDR3 length diversity within CD45RA and CD45RO cells (26,
27). For example, in analyses of V families within CD8+ CD45RO cells, in which predominant peaks composed over
80% of the area under the curve (Fig. 3 and 4), there was no evidence of contamination within the CD45RA PCR products. Similarly, multiple peaks seen within CD45RA T cells failed to contaminate the predominant cDNA of the CD8+ CD45RO T cells. The considerable degree of
resolution of predominant peaks within CD45RA and CD45RO T cells
greatly enhanced our analysis of perturbations within the TCR repertoire.
The CDR3 lengths within both the CD45RA and CD45RO T-cell subsets from
healthy children and neonates demonstrated a Gaussian distribution. In
contrast, we easily detected oligoclonal expansions within
CD8+ CD45RO cells that were concealed when unfractionated
CD8+ T cells from HIV-infected patients were examined.
Patterns of CD8+ T-cell clonal dominance have previously
been observed in HIV-infected children on the basis of an
evaluation of unfractionated CD8+ T cells (30).
Analysis of highly enriched CD45RA and CD45RO CD8+ T
cells indicates that clonal predominance may be underestimated if
the analysis is limited to unfractionated cells. The CD8+
CD45RO T cells from all four of the HIV-infected children whose cells were examined displayed evidence of oligoclonal expansion. Most
importantly, through the enrichment for the CD45 subpopulations and precise quantitation of CDR3 size diversity, we were able to
accurately measure changes that occur within the TCR repertoire during
the course of HIV infection.
We easily amplified, cloned, and sequenced CDR3 regions from Jurkat
cells and from lymphocytes in cord blood or PBMC. In all cases sequence
analysis confirmed the spectratyping findings. All clones from Jurkat
cells carried the same amino acid sequences. Polyclonal cord blood or
PBMC T cells that had CDR3 lengths which conformed to a Gaussian
distribution consisted of cells with a mixture of sequences. Most
significantly, a predominant CDR3 peak in CD8+ CD45RO T
cells from HIV-infected individuals represented an oligoclonal expansion of a specific TCR clonotype by sequence analysis. These results indicate that spectratyping can be used as an accurate surrogate marker to assess the extent of clonal diversity in healthy and immune-suppressed individuals. We have shown that when TCR CDR3
spectratyping is applied to distinct subpopulations of T cells it can
provide a high degree of insight into both the breadth and the
specificity of the T-cell immune response. When stringent amplification
conditions, precise primers, and effective cell separation techniques
are applied together, the diversity of the T-cell immune response can
be accurately measured in both health and disease states.
| |
ACKNOWLEDGMENTS |
We thank Willam G. Farmerie, Dan Brazeau, and Ginger Clark for
help with the GeneScan analysis.
This work was supported by Public Health Service awards RO1
HD32259 and RO1 HL58005, National Institutes of
Health-sponsored General Clinical Research Center grant
RR0082, an award from the Elizabeth Glazer Pediatric AIDS Foundation
(grant PG-50956), and The University of Florida Interdisciplinary
Center for Biological Research.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pediatrics, Division of Immunology and Infectious Diseases, College of Medicine, University of Florida, Box 100296, 1600 S.W. Archer Rd.,
Gainesville, FL 32610-0296. Phone: (352) 392-2961. Fax: (352) 392-0481. E-mail: Sleasjw{at}peds.ufl.edu.
| |
REFERENCES |
| 1.
|
Akbar, A. N.,
L. Terry,
A. Timms,
P. C. Beverley, and G. Janossy.
1988.
Loss of CD45R and gain of UCHL1 reactivity is a feature of primed T cells.
J. Immunol.
140:2171-2178[Abstract].
|
| 2.
|
Aleixo, L. F.,
M. M. Goodenow, and J. W. Sleasman.
1995.
Molecular analysis of highly enriched populations of T-cell-depleted monocytes.
Clin. Diagn. Lab. Immunol.
2:733-739[Abstract].
|
| 3.
|
Brownstein, M. J.,
J. D. Carpten, and J. R. Smith.
1996.
Modulation of non-templated nucleotide addition by Taq DNA polymerase: primer modifications that facilitate genotyping.
BioTechniques
20:1004-1010[Medline].
|
| 4.
|
Callan, M. F.,
N. Steven,
P. Krausa,
J. D. Wilson,
P. A. Moss,
G. M. Gillespie,
J. I. Bell,
A. B. Rickinson, and A. J. McMichael.
1996.
Large clonal expansions of CD8+ T cells in acute infectious mononucleosis.
Nat. Med.
2:906-911[CrossRef][Medline].
|
| 5.
|
Chen, Z. W.,
Z. C. Kou,
C. Lekutis,
L. Shen,
D. Zhou,
M. Halloran,
J. Li,
J. Sodroski,
D. Lee-Parritz, and N. L. Letvin.
1995.
T cell receptor V beta repertoire in an acute infection of rhesus monkeys with simian immunodeficiency viruses and a chimeric simian-human immunodeficiency virus.
J. Exp. Med.
182:21-31[Abstract/Free Full Text].
|
| 6.
|
Chen, Z. W.,
Z. C. Kou,
L. Shen,
J. D. Regan,
C. I. Lord,
M. Halloran,
D. Lee-Parritz,
P. N. Fultz, and N. L. Letvin.
1994.
An acutely lethal simian immunodeficiency virus stimulates expansion of V beta 14-expressing T lymphocytes.
Proc. Natl. Acad. Sci. USA
91:7501-7505[Abstract/Free Full Text].
|
| 7.
|
Chen, Z. W.,
Z. C. Kou,
L. Shen,
K. A. Reimann, and N. L. Letvin.
1993.
Conserved T-cell receptor repertoire in simian immunodeficiency virus-infected rhesus monkeys.
J. Immunol.
151:2177-2187[Abstract].
|
| 8.
|
Clement, L. T.
1992.
Isoforms of the CD45 common leukocyte antigen family: markers for human T-cell differentiation.
J. Clin. Immunol.
12:1-10[CrossRef][Medline].
|
| 9.
|
Clement, L. T.,
N. Yamashita, and A. M. Martin.
1988.
The functionally distinct subpopulations of human CD4+ helper/inducer T lymphocytes defined by anti-CD45R antibodies derive sequentially from a differentiation pathway that is regulated by activation-dependent post-thymic differentiation.
J. Immunol.
141:1464-1470[Abstract].
|
| 10.
|
Connors, M.,
J. A. Kovacs,
S. Krevat,
B. J. Gea,
M. C. Sneller,
M. Flanigan,
J. A. Metcalf,
R. E. Walker,
J. Falloon,
M. Baseler,
I. Feuerstein,
H. Masur, and H. C. Lane.
1997.
HIV infection induces 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.
3:533-540[CrossRef][Medline].
|
| 11.
|
De Paoli, P.,
S. Battistin, and G. F. Santini.
1988.
Age-related changes in human lymphocyte subsets: progressive reduction of the CD4 CD45R (suppressor inducer) population.
Clin. Immunol. Immunopathol.
48:290-296[CrossRef][Medline].
|
| 12.
|
Fields, B. A.,
E. L. Malchiodi,
H. Li,
X. Ysern,
C. V. Stauffacher,
P. M. Schlievert,
K. Karjalainen, and R. A. Mariuzza.
1996.
Crystal structure of a T-cell receptor beta-chain complexed with a superantigen.
Nature
384:188-192[CrossRef][Medline].
|
| 13.
|
Fitzgerald, J. E.,
N. S. Ricalton,
A. C. Meyer,
S. G. West,
H. Kaplan,
C. Behrendt, and B. L. Kotzin.
1995.
Analysis of clonal CD8+ T cell expansions in normal individuals and patients with rheumatoid arthritis.
J. Immunol.
154:3538-3547[Abstract].
|
| 14.
|
Garcia, K. C.,
M. Degano,
R. L. Stanfield,
A. Brunmark,
M. R. Jackson,
P. A. Peterson,
L. Teyton, and I. A. Wilson.
1996.
An alphabeta T cell receptor structure at 2.5 Å and its orientation in the TCR-MHC complex.
Science
274:209-219[Abstract/Free Full Text].
|
| 15.
|
Gea-Banacloche, J. C.,
E. E. Weiskopf,
C. Hallahan,
J. C. Lopez Bernaldo de Quiros,
M. Flanigan,
J. M. Mican,
J. Falloon,
M. Baseler,
R. Stevens,
H. C. Lane, and M. Connors.
1998.
Progression of human immunodeficiency virus disease is associated with increasing disruptions within the CD4+ T cell receptor repertoire.
J. Infect. Dis.
177:579-585[Medline].
|
| 16.
|
George, J. F., Jr., and H. W. Schroeder, Jr.
1992.
Developmental regulation of D beta reading frame and junctional diversity in T cell receptor-beta transcripts from human thymus.
J. Immunol.
148:1230-1239[Abstract].
|
| 17.
|
Gorochov, G.,
A. U. Neumann,
A. Kereveur,
C. Parizot,
T. Li,
C. Katlama,
M. Karmochkine,
G. Raguin,
B. Autran, and P. Debre.
1998.
Perturbation of CD4+ and CD8+ T-cell repertoires during progression to AIDS and regulation of the CD4+ repertoire during antiviral therapy.
Nat. Med.
4:215-221[CrossRef][Medline].
|
| 18.
|
Grunewald, J., and H. Wigzell.
1999.
T cell receptors in health and disease. Introduction.
Springer Semin. Immunopathol.
21:1-4[Medline].
|
| 19.
|
Halapi, E.,
M. Jeddi-Tehrani,
A. Blucher,
R. Andersson,
P. Rossi,
H. Wigzell, and J. Grunewald.
1999.
Diverse T-cell receptor CDR3 length patterns in human CD4+ and CD8+ T lymphocytes from newborns and adults.
Scand. J. Immunol.
49:149-154[CrossRef][Medline].
|
| 20.
|
Kostense, S.,
F. M. Raaphorst,
D. W. Notermans,
J. Joling,
B. Hooibrink,
N. G. Pakker,
S. A. Danner,
J. M. Teale, and F. Miedema.
1998.
Diversity of the T-cell receptor BV repertoire in HIV-1-infected patients reflects the biphasic CD4+ T-cell repopulation kinetics during highly active antiretroviral therapy.
AIDS
12:F235-F240[CrossRef][Medline].
|
| 21.
|
Kou, Z. C.,
M. Halloran,
D. Lee-Parritz,
L. Shen,
M. Simon,
P. K. Sehgal,
Y. Shen, and Z. W. Chen.
1998.
In vivo effects of a bacterial superantigen on macaque TCR repertoires.
J. Immunol.
160:5170-5180[Abstract/Free Full Text].
|
| 22.
|
Posnett, D. N.,
R. Sinha,
S. Kabak, and C. Russo.
1994.
Clonal populations of T cells in normal elderly humans: the T cell equivalent to "benign monoclonal gammapathy."
J. Exp. Med.
179:609-618[Abstract/Free Full Text]. (Erratum, 179:1077.)
|
| 23.
|
Prochnicka-Chalufour, A.,
J. L. Casanova,
S. Avrameas,
J. M. Claverie, and P. Kourilsky.
1991.
Biased amino acid distributions in regions of the T cell receptors and MHC molecules potentially involved in their association.
Int. Immunol.
3:853-864[Abstract/Free Full Text].
|
| 24.
|
Raaphorst, F. M.,
E. L. Kaijzel,
M. J. van Tol,
J. M. Vossen, and P. J. van den Elsen.
1994.
Non-random employment of V beta 6 and J beta gene elements and conserved amino acid usage profiles in CDR3 regions of human fetal and adult TCR beta chain rearrangements.
Int. Immunol.
6:1-9[Abstract/Free Full Text].
|
| 25.
|
Schwab, R.,
P. Szabo,
J. S. Manavalan,
M. E. Weksler,
D. N. Posnett,
C. Pannetier,
P. Kourilsky, and J. Even.
1997.
Expanded CD4+ and CD8+ T cell clones in elderly humans.
J. Immunol.
158:4493-4499[Abstract].
|
| 26.
|
Sleasman, J. W.,
L. F. Aleixo,
A. Morton,
S. S. Skoda, and M. M. Goodenow.
1996.
CD4+ memory T cells are the predominant population of HIV-1-infected lymphocytes in neonates and children.
AIDS
10:1477-1484[Medline].
|
| 27.
|
Sleasman, J. W.,
T. O. Harville,
G. B. White,
J. F. George,
D. J. Barrett, and M. M. Goodenow.
1994.
Arrested rearrangement of TCR V beta genes in thymocytes from children with X-linked severe combined immunodeficiency disease.
J. Immunol.
153:442-448[Abstract].
|
| 28.
|
Sleasman, J. W.,
B. H. Leon,
L. F. Aleixo,
M. Rojas, and M. M. Goodenow.
1997.
Immunomagnetic selection of purified monocyte and lymphocyte populations from peripheral blood mononuclear cells following cryopreservation.
Clin. Diagn. Lab. Immunol.
4:653-658[Abstract].
|
| 29.
|
Tedder, T. F.,
L. T. Clement, and M. D. Cooper.
1985.
Human lymphocyte differentiation antigens HB-10 and HB-11.
J. Immunol.
134:2983-2988[Abstract].
|
| 30.
|
Than, S.,
M. Kharbanda,
V. Chitnis,
S. Bakshi,
P. K. Gregersen, and S. Pahwa.
1999.
Clonal dominance patterns of CD8 T cells in relation to disease progression in HIV-infected children.
J. Immunol.
162:3680-3686[Abstract/Free Full Text].
|
| 31.
|
Toyonaga, B.,
Y. Yoshikai,
V. Vadasz,
B. Chin, and T. W. Mak.
1985.
Organization and sequences of the diversity, joining, and constant region genes of the human T-cell receptor beta chain.
Proc. Natl. Acad. Sci. USA
82:8624-8628[Abstract/Free Full Text].
|
| 32.
|
Wilson, J. D.,
G. S. Ogg,
R. L. Allen,
P. J. Goulder,
A. Kelleher,
A. K. Sewell,
C. A. O'Callaghan,
S. L. Rowland-Jones,
M. F. Callan, and A. J. McMichael.
1998.
Oligoclonal expansions of CD8(+) T cells in chronic HIV infection are antigen specific.
J. Exp. Med.
188:785-790[Abstract/Free Full Text].
|
Clinical and Diagnostic Laboratory Immunology, November 2000, p. 953-959, Vol. 7, No. 6
1071-412X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Sarzotti, M., Patel, D. D., Li, X., Ozaki, D. A., Cao, S., Langdon, S., Parrott, R. E., Coyne, K., Buckley, R. H.
(2003). T Cell Repertoire Development in Humans with SCID After Nonablative Allogeneic Marrow Transplantation. J. Immunol.
170: 2711-2718
[Abstract]
[Full Text]
Top
Abstract
Introduction
