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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 3 (February 1), 1998:
pp. 949-955
Development of Autologous, Oligoclonal, Poorly Functioning T
Lymphocytes in a Patient With Autosomal Recessive Severe Combined
Immunodeficiency Caused by Defects of the Jak3 Tyrosine Kinase
By
Duilio Brugnoni,
Luigi D. Notarangelo,
Alessandra Sottini,
Paolo Airò,
Marta Pennacchio,
Evelina Mazzolari,
Simona Signorini,
Fabio Candotti,
Anna Villa,
Patrizia Mella,
Paolo Vezzoni,
Roberto Cattaneo,
Alberto G. Ugazio, and
Luisa Imberti
From the Servizio di Immunologia Clinica, Clinica Pediatrica,
Consorzio per le Biotecnologie, III Laboratorio Analisi, Spedali
Civili, Brescia; and Istituto di Tecnologie Biomediche Avanzate, CNR,
Milano, Italy.
 |
ABSTRACT |
Defects of the common gamma chain subunit of the cytokine receptors
( c) or of Jak3, a tyrosine kinase required for
c signal transduction, result in
T B+ severe combined immunodeficiency
(SCID). However, atypical cases, characterized by progressive
development of T lymphocytes, have been also reported. We describe a
child with SCID caused by Jak3 gene defects, which strongly but not
completely affect Jak3 protein expression and function, who developed a
substantial number (>3,000/µL) of autologous
CD3+CD4+ T cells. These cells showed a
primed/activated phenotype (CD45R0+ Fas+
HLA-DR+ CD62Llo), defective secretion of
T-helper 1 and T-helper 2 cytokines, reduced proliferation to mitogens,
and a high in vitro susceptibility to spontaneous (caused by
downregulation of bcl-2 expression) as well as activation-induced cell
death. A restricted T-cell receptor repertoire was observed, with
oligoclonal expansion within each of the dominant segments. These
features resemble those observed in
c-/y and in Jak3/
mice, in which a population of activated, anergic T cells
(predominantly CD4+) also develops with age. These
results suggest that residual Jak3 expression and function or other
Jak3-independent signals may also permit the generation of
CD4+ T cells that undergo in vivo clonal expansion in
humans; however, these mechanisms do not allow development of
CD8+ T cells, nor do they fully restore the functional
properties of CD4+ T lymphocytes.
| |
INTRODUCTION |
JAK3, A MEMBER of the Janus family
tyrosine kinase,1,2 is an intracellular kinase
required for signaling through the common gamma chain
(c),3-5 a subunit of the cytokine receptors for interleukin-2 (IL-2), IL-4, IL-7, IL-9, and IL-15.6
Functional integrity of the c/Jak3 system is essential
for lymphoid development,7-9 signaling,10 and
survival.11 In humans, genetic defects affecting the IL-2RG
gene (encoding for c) or the Jak3 gene result in
X-linked12-14 or in autosomal recessive15-19 severe combined immunodeficiency (XL-SCID or AR-SCID), respectively, with lack of circulating T cells and a normal or increased number of B
lymphocytes that are, however, functionally defective.20
Disruption of the IL-2RG or the Jak3 genes in mice also results in
SCID, but with a distinct biological phenotype, characterized by a
severe defect of T-, B-, and natural killer (NK)-cell differentiation, that allows an age-dependent increase of activated and anergic CD4+ T cells in the periphery.13,14,17-19 Jak3
protein expression in the thymus is essential to reconstitute thymocyte
development (including  and NK cells); furthermore, maintenance
of peripheral expression of Jak3 is required to correct the
immunological abnormalities (T-cell anergy) observed in
Jak3/ mice.21
In humans, atypical cases of XL-SCID, characterized by development of T
lymphocytes, have been recently described.4,22-24 With the
exception of the case reported by Stephan et al, in which development
of autologous T lymphocytes resulted from a spontaneous reversion of
the genetic defect at the IL-2RG gene locus,24 T cells
remained genetically and functionally defective in other patients with
atypical XL-SCID.4,22,23
We report the case of an infant with
TB+ SCID caused by Jak3
mutations, which strongly but not completely affect Jak3
protein expression and function, who developed early in life a
substantial number of autologous, activated, and oligoclonal
CD3+CD4+ T cells that remained genetically and
functionally defective.
| |
MATERIALS AND METHODS |
Case report.
The infant is the only son of nonconsanguineous parents. The diagnosis
of SCID was serendipitously obtained at birth, when profound
abnormalities of lymphocyte subpopulations
(Table 1) were disclosed during blood
testing for an episode of intestinal subobstruction that resolved
spontaneously at 1 week of age. The infant was admitted to the Bone
Marrow Transplantation Unit of the Department of Pediatrics, University
of Brescia, at 1 month of age. At that time, chest radiograph
examination revealed mild interstitial pneumonia. He was hospitalized
in a laminar flow unit, and he was treated with intravenous
immunoglobulins, antibiotics, and acyclovir until the age of 3 months,
when he received haploidentical bone marrow transplantation (BMT) with
T-depleted marrow from the father following conditioning with busulphan
and cyclophosphamide. Before BMT, an increase of T-cell count was
detected in the periphery (Table 1).
The molecular defect in the Jak3 gene and the biochemical analysis of
the Jak/STAT signaling pathway have been reported in detail elsewhere,
together with other Jak3-deficient SCID patients (patient
LE25). Briefly, the patient carries distinct mutations on
the two Jak3 alleles: an intragenic deletion that results in several
mutant splicing products, including an in-frame deleted cDNA form
lacking exons 10 to 12, and a de novo A to G transition at the
nucleotide 1537, leading to a glutamic acid to glycine substitution at
codon 481 (E481G). Both mutant forms of the Jak3 protein were expressed in lymphoblastoid cell lines, albeit in trace amounts, as detected by
Western blotting. Furthermore, residual preservation of the function of
the E481G Jak3 mutant was shown, based on reduced but detectable
phosphorylation of Jak3 and STAT5 proteins in response to
IL-2.25 Molecular analysis of the patient's peripheral
blood T cells revealed that they were autologous (as detected by highly polymorphic DNA markers D1S80 and DQ alpha) and genetically defective, as shown by mutation analysis at the Jak3 locus (data not shown).
After BMT, the patient developed hemolytic anemia, associated with the
presence of autologous and donor-derived T cells in the periphery.
Lymphocyte surface phenotype analysis.
Evaluation of lymphocyte surface membrane expression was performed on
heparinized whole blood samples with the following monoclonal antibodies (MoAbs): fluorescein-isothiocyanate (FITC)-conjugated OKT3/CD3, FITC-OKT8/CD8, FITC- or phycoerytrin (PE)-conjugated OKT4/CD4, PE-OKDR/HLA DR (Ortho, Raritan, NJ), PE-Leu11c/CD16, PE-Leu45RO/CD45RO (Becton Dickinson, Mountain View, CA), FITC-B4/CD19, PE-2H4/CD45RA (Coulter Immunology, Hialeah, FL), PE-DREG-56/CD62L, and
FITC-DX2/CD95 (Pharmingen, San Diego, CA).
After two washes in phosphate-buffered saline (PBS)-1% fetal calf
serum (FCS), samples were analyzed with a flow cytometer equipped with
an argon-ion laser (488 nm; Cytoron Absolute, Ortho), using the ABS
software. Gates were selected on lymphoid cells as determined by
forward and right-angle scatter properties; dead cells were excluded by
setting an appropriate threshold trigger on the low forward light
scatter parameter; and nonspecific staining was assessed using
FITC-conjugated nonimmune mouse IgG (Coulter). Data were acquired and
stored in list mode. Fluorescence analysis was performed on a log
scale.
Preparation of lymphocytes.
Mononuclear cells (PBMC) were separated from the peripheral blood by
Ficoll-Hypaque (Pharmacia Biotech, Piscataway, NJ) density gradient
centrifugation and washed three times in RPMI-1640 (GIBCO Laboratories,
Grand Island, NY).
Lymphocyte activation.
All experiments were performed in RPMI-1640 medium supplemented with
10% heat-inactivated FCS, 1% glutamin, and antibiotics. Proliferative
responses were measured in triplicates of 200 µL containing 1 × 105 total PBMC using various combinations of the following
stimulating agents: immobilized anti-CD3 (OKT3) MoAb, 200 ng/mL, with
or without IL-2, 20 U/mL (Biosource International, Camarillo, CA);
phytoemoagglutinin (PHA), 1.2 µg/mL (Irvine Scientific, Santa Ana,
CA); phorbol 12-myristate 13-acetate (PMA), 5 ng/mL (Sigma, St Louis,
MO) plus ionomycin, 500 ng/mL (Sigma); and 1 × 105
irradiated (2,000 rads) allogeneic PBMC from a pool of healthy donors.
After 72 hours of culture (or 120 hours for the mixed lymphocyte
culture) at 37°C, 5% CO2, the cells were pulsed
overnight with 1 µCi (3H)-thymidine and were obtained
subsequently. Incorporated radioactivity was measured by liquid
scintillation counting (Beckman, Fullerton, CA), and the results were
expressed as mean counts per minute of triplicates.
Apoptosis assay.
PBMC, cultured with or without the stimulating agents in the conditions
indicated above, were also evaluated for apoptosis.
After 72 hours of culture, cells were washed and the percentage of
hypodiploid nuclei (less than diploid DNA content) was determined with
a slight modification of the method of Nicoletti et al,26
as described.27 Briefly, 1 × 106 PBMC
were fixed in 1 mL of cold 70% ethanol at 4°C for 20 minutes. Cells were then washed, incubated at room temperature for 1 minute with
RNase (0.5 mg/mL; Boehringer GmbH, Mannheim, Germany), for 15 minutes
with propidium iodide (100 mg/mL; Sigma), and immediately analyzed. The
correct threshold was selected experimentally using the model system of
apoptosis induced in murine thymocytes by 72 hours culture with
dexamethasone 107 mol/L. Apoptotic cell nuclei, easily
distinguishable from debris owing to the condensation of nuclear
chromatin, emitted red fluorescence in the RD-FL channels 46 to 146. As
there was no overlap between apoptotic nuclei and debris, the small
percentage of residual low-fluorescence detritus (<1% in normal
cells) was eliminated by gating the red fluorescence scale at channel
45. All measurements were performed with the same instrument setting.
Analysis of intracellular bcl-2 expression.
A total of 5 × 105 PBMC were permeabilized using
Permeafix (Ortho) for 40 minutes, before an incubation with a rabbit
polyclonal antibody to bcl-2 (Santa Cruz Biotechnology Inc, Santa Cruz,
CA), or with polyclonal nonimmune rabbit IgG. After 30 minutes, cells were washed and incubated with FITC-conjugated goat anti-rabbit IgG
antibody (Santa Cruz Biotechnology Inc). Quantitation of positivity for
bcl-2 was performed using QuickCal beads (Flow Cytometry Standards Corp, San Juan, PR), and results were expressed as specific molecule equivalents of soluble fluorochrome (MESF), after subtracting the
values obtained for nonspecific fluorescence (always <2,000 MESF).
Cytokine production and measurement.
For cytokine production, 1 × 106 PBMC were
cultured at 37°C, 5% CO2 in 24-well flat-bottomed
plates in a final volume of 1 mL with PMA (5 ng/mL) plus ionomycin (500 ng/mL). After 72 hours, supernatants were collected and frozen at
 20°C until use. Sandwich enzyme-linked immunosorbent assay
(ELISA) commercial kits were used to measure IL-2 (Genzyme, Cambridge,
MA), IL-4 (BioSource International), IL-5 (CytImmune Sciences, College
Park, MD), and interferon (IFN)- (BioSource International).
Analysis of the T-cell receptor repertoire diversity.
One microgram of the total RNA, prepared with the guanidinium
thiocyanate-phenol-chloroform method from PBMC, was used to synthesize
the first strand of TCRB chain cDNA using a specific TCRBC primer
( cDNA: 5 GGG CTG CTC CTT GAG GGG CTG CGG 3) and the
RiboClone cDNA Synthesis System (Promega Corporation, Madison, WI).
cDNA was subjected to enzymatic amplification using a second human
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). Forty cycles of
polymerase chain reaction (PCR) were performed under the following
conditions: denaturation at 94°C for 1 minute, annealing at
52°C for 1 minute, and extension at 72°C for 1 minute; the last
cycle extension was performed at 72°C for 7 minutes. The
specificity of the total amplified products was analyzed using a
colorimetric method and biotinylated TCRBV-specific
probes.28 Subsequently, the TCRBV chains of interest were
amplified by 35 cycles of PCR, using TCRBV-specific family primers
(TCRBV2: 5 TCA TCA ACC ATG CAA GCC TGA CCT 3; TCRBV4:
5 GCC CAA ACC TAA CAT TCT CAA CTC 3; TCRBV5S1: 5
ATA CTT CAG TGA GAC ACA GAG AAA 3; TCRBV6: 5 AGG CCT GAG
GGA TCC GTC TC 3; and TCRBV14: 5 GTC TCT CGA AAA GAG AAG
AGG AAT 3) and the TCRBC oligonucleotide AI. For heteroduplex
analysis, 20 µL of TCRBV-specific products were heated to 95°C
for 5 minutes and cooled to 50°C for 1 hour. The annealed samples
were kept on ice and then run for 5 to 6 hours at 200 V, at room
temperature, on a 12% nondenaturing 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). DNA Molecular Weight V
(Boehringer Mannheim) was used as size marker. The gel was 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
photographed under ultraviolet light. TCRBV8-specific amplified
products from the T-cell line J77 and from PBMC stimulated with an
anti-TCRBV8 MoAb were used as monoclonal and polyclonal controls,
respectively.29 TCRBV2 and TCRBV5S1 products, obtained from
a new amplification performed with specific primers, were purified,
cloned, and sequenced as recently described.30 Sequences
were compared with published data relative to TCRBV, TCRBD, TCRBJ, and
TCRBC segments.31,32
| |
RESULTS |
Immunophenotypical and molecular studies.
Cytofluorimetric analysis performed on PBMC collected from the
patient at the time of birth revealed a typical
TB+ SCID phenotype, with a severely
reduced proportion of peripheral blood T lymphocytes and an increased
proportion of B cells (Table 1).
Interestingly, at 3 months of age the patient developed autologous (as
assessed by HLA-typing) CD3+CD4+ cells that
increased to 41% just before undergoing haploidentical BMT from his
father (Table 1). However, these lymphocytes were phenotypically
abnormal, as they predominantly coexpressed the activation marker
HLA-DR, were almost exclusively CD45R0+ and
CD95/Fas+, and showed a reduced density of CD62L
(Fig 1). These features are typical of
primed/activated T cells.
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| Fig 1.
Two-color analysis of activation/memory markers
expression by peripheral CD4+ lymphocytes from the
patient at 3 months of age and from one representative age-matched
healthy control. Data are presented as relative log fluorescence
intensity.
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Normal expression of the c was found on the lymphocyte
surface, as detected with the rat TUGh4 MoAb (a kind gift of
Dr K. Sugamura, Department of Microbiology, Tohoku
University School of Medicine, Japan; data not shown).
TCRBV repertoire diversity.
The analysis of the TCRBV usage was performed on patient's PBMC at 3 months of age and compared with that of age-matched healthy controls.
Figure 2A shows that all TCRBV transcripts,
with the only exception of TCRBV13S1 chain, can be amplified from the
patient's lymphocytes without evidence for over-representation of
single TCRBV population. On the contrary, the most notable difference is the greatly reduced percentage of TCRBV3, TCRBV13S1, and TCRBV13S2 chains in the patient as compared with controls. However, because we
have previously found that the level of TCRBV diversity is not
necessarily related to the relative percentage of TCRBV
expression,33 we also performed a heteroduplex analysis of
TCRBV2, TCRBV4, TCRBV5S1, TCRBV6, and TCRBV14 transcripts, which were
expressed at highest levels in the patient's T cells. This technique
is based on the different ability of amplified TCRBV segments to
migrate in a polyacrylamide gel, depending on their monoclonal or
polyclonal composition.29 As shown in Fig 2B, all the PCR
products obtained with the 5 TCRBV family-specific primers generated
homoduplex bands that are indicative of predominant homogeneous
molecular species in the context of a background of heteroduplex bands
or of smears, respectively, representing minor oligoclonal or
polyclonal expansions. On the contrary, the amplified products of the
same TCRBV segments prepared from PBMC of an age-matched healthy
control and chosen among those analyzed in Fig 2A generated smears or faint heteroduplex bands. This result is consistent with our previous observations suggesting that, in the absence of antigenic stimulation, most of the TCRBV segments expressed by PBMC from normal individuals are largely polyclonal.34
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| Fig 2.
(A) TCRBV usage in patient with AR-SCID ( ) and in
healthy controls ( ). The relative percentage of TCRBV segments
expression was calculated by normalizing the OD value of each
individual TCRBV segment with respect to the sum of the OD values of
all the 26 TCRBV chains as follows: % of expression:
(B)
Heteroduplex analysis of the indicated TCRBV chains prepared from
patient's and control's lymphocytes after PCR amplification performed
with TCRBV-specific primers. TCRBV8 amplified products, obtained from
the amplification of the J77 and C1-632 cells RNA29 were
used as monoclonal and polyclonal controls and loaded, respectively, in
the first and second lane of the gel. MW; molecular weight marker. (C)
Junctional amino acid TCRBV2 and TCRBV5S1 sequences were deduced from
nucleotide sequences and displayed as standard one-letter code. Only
the last 3 amino acids of the TCRBV segments and the first
5 amino acids of TCRBJ chains are shown.
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The strongest evidence for oligoclonal expansion that confirmed the
results obtained with the heteroduplex analysis was provided by
sequencing of TCRBV2 and TCRBV5S1 transcripts. In fact, in both groups
of sequences the dominance of a single clone was detected (Fig 2C). We
did not observe, however, amino acid similarities in the NDN regions of
the two major groups of clones.
Functional studies.
In accordance with the lack of T cells at birth, in vitro response of
PBMC to T-cell activating agents (PHA, anti-CD3, and anti-CD3 plus
IL-2) was abolished, whereas stimulation with PMA plus ionomycin, which
can also elicit B-cell activation, resulted in a substantial
proliferative response (Table 2).
A defect of the proliferative response to PHA, anti-CD3, and pooled
unrelated cells was also observed after appearance of circulating
autologous T lymphocytes. This defect was too profound to be explained
solely by the reduction of the T-cell number as compared with the
controls' (Table 2). A moderate increase of proliferation was obtained
after the addition of exogenous IL-2 to both unstimulated and
stimulated cultures, in keeping with the strongly reduced, but
detectable, levels of Jak3 and STAT5 phosphorylation observed after the
stimulation with IL-2.25 These observations suggested a
defective IL-2 production following in vitro culture with the
activating stimuli mentioned above.
In fact, even under powerful stimulating conditions (PMA plus ionomycin
for 72 hours), patient PBMC produced markedly lower levels of IL-2 and
IFN- than in the normal controls (Fig
3). On the other hand, no increase in the production of IL-4 and IL-5 was observed (Fig 3), thus ruling out the possibility that the defective proliferative response was accounted for by a shift toward
Th2-type cytokine secreting cells. In view of the substantial number of
T cells in the periphery, this defect was also too profound to be
explained solely by the reduction of the T-cell number as compared with
the controls'.
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| Fig 3.
Cytokine production by total PBMC from the patient at the
age of 3 months () and from 10 age-matched healthy controls ( ) in
response to PMA (5 ng/mL) plus ionomycin (500 ng/mL). Data of healthy
controls are expressed as mean ± 1 SD.
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We considered the possibility that defective lymphocyte proliferation
and the reduced cytokine production were caused by increased cell death
during the culture. Indeed, an increased proportion of apoptotic cells
was detected in the patient, both in unstimulated and stimulated
cultures (Fig 4).
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| Fig 4.
Increased rates of apoptotic cells in unstimulated and
stimulated cultures of PBMC of the patient () compared with 10 age-matched healthy controls (). Data of healthy controls are
expressed as mean ± 1 SD. All cultures were performed for 72 hours.
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Because the products of bcl-2 gene are known to modulate the
susceptibility of T cells to spontaneous apoptosis,11
cytoplasmic levels of this protein were evaluated by flow cytometry.
The intensity of staining for bcl-2, expressed as MESF, was
significantly reduced after 48 hours of unstimulated culture of
lymphocytes from the patient, as compared with that observed in three
age-matched healthy controls (4,592 v 14,750 ± 555;
Fig 5). Taken together, these data suggest
that the raised number of spontaneously dying PBMC in the patient is
accounted for by downmodulation of bcl-2 gene product expression.
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| Fig 5.
The expression of bcl-2 on PBMC from the patient
(continuous lines) and one representative healthy control (dashed
lines) after 48 hours of unstimulated culture.
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| |
DISCUSSION |
We report a case of a child with an AR-SCID, caused by Jak3 mutations,
presenting at birth a typical TB+
phenotype, who developed autologous CD3+CD4+ T
cells after the first few months of life. These lymphocytes showed a
primed/activated phenotype; had a severely defective proliferative
response to anti-CD3, PHA, and allogeneic cells; and secreted very low
levels of Th1 and Th2 cytokines, as compared with controls. Functional
defects, including reduced proliferative response and defective
cytokine production, were accounted for by the large number of cells
dying either spontaneously (as a consequence of bcl-2 downregulation)
or after activation in vitro. These data are in keeping with the notion
that the response induced in T lymphocytes by antigen stimulation is
influenced by their activation status, because triggering of TCR/CD3
complex of recently activated lymphocytes leads to apoptosis rather
than proliferation.35-37
Our report is similar to other atypical presentations of XL-SCID, in
which the presence of autologous, poorly functioning T lymphocytes was
documented.4,22-24 In particular, the patient reported by
DiSanto et al22 carried a splice site mutation in the
c gene leading to two alternative transcripts, only one
being functional, although present in limited amounts. Furthermore, Russell et al4 reported another XL-SCID patient with a
missense mutation in the cytoplasmic region of the c
protein that substantially diminished, but did not abrogate, its
association with Jak3. In this latter case, the patient at birth had no
T lymphocytes and developed CD3+CD4+ cells at
the age of 19 weeks.38 These results suggest that reduced
but detectable expression of c4,22 or Jak3
(present case) proteins may be enough to generate a limited number of
peripheral T-cell clones. Alternatively, other hematopoietic growth
factors that do not use Jak3 (eg, IL-3, Thymic Stromal Lymphopoietin,39 or stem cell factor through its receptor
c-kit40) might substitute for some of the functions of
Jak3-dependent cytokines allowing the generation of T cells. However,
in the absence of c/Jak3 signals the action of these
cytokines might be insufficient for the correct activation and function
of these lymphocytes. In particular, IL-7 receptor signaling has been
recently shown to upregulate bcl-2 expression in developing thymocytes
and in mature peripheral T cells,41,42 thus maintaining
their viability and function.41,42
Interestingly, murine models of defective Jak3 function
(Jak3/ mice) also have a similar, abnormal
T-cell development. T-cell differentiation in the thymus is partially
conserved, despite severe cell depletion. Furthermore, an age-dependent
increase of CD4+ lymphocytes is observed in the periphery.
These cells have an activated/memory phenotype, fail to respond to
mitogens, and show a reduced IL-2 secretion following stimulation with
anti-CD3 plus anti-CD28.17-19
The observation that, like in Jak3/ mice,
preferential accumulation of CD4+ lymphocytes was observed
in our patient suggests that cytokine signaling through the
c/Jak3 pathway may be more critical for the maturation
or survival of CD8 than of the CD4 lineage T cells.
In our experience, oligoclonal, in vivo-activated, poorly functioning T
cells were observed in other immunodeficiencies. In Omenn's syndrome,
a rare inherited immunodeficiency, peripheral blood T cells, usually
present in normal number, are oligoclonal43 and show an
activated phenotype and a defective proliferative response to
mitogens.44 As in the case described here, we have recently
shown that functional T-cell defects observed in Omenn's syndrome
might be explained, at least in part, by excessive cell death resulting
from two main mechanisms: spontaneous apoptosis in unstimulated
cultures, associated with reduced expression of bcl-2 gene product, and
susceptibility to activation-induced cell death through a Fas/Fas
ligand-mediated mechanism, after restimulation in vitro with
mitogens.45 Activated, anergic T cells have been also
detected in combined immunodeficiency caused by functional defects
(unpublished observation, February 1997), in early phases of immune reconstitution following BMT,46-48 and in
trans-placental engraftment of maternal T cells occurring in some cases
of SCID TB+/.49
In all cases, the unknown developmental pathways used by T lymphocytes
seem to be antigen driven; furthermore, they lead to terminally
differentiated T cells that are functionally defective because of high
susceptibility to undergo cell death.
On the other hand, not much is known about the origin of these
activated and anergic T lymphocytes. Refilling of a depleted T-cell
compartment may arise from two main potential ways: thymopoiesis, which
creates new T cells from progenitors and maintains or increases the
potential diversity of T-cell repertoire, and peripheral expansion, which probably restricts that repertoire. It is unlikely that defective
thymopoiesis alone may account for the aberrant T-cell phenotype
observed in our patient, as in this case accumulation of
"naïve" T cells would be expected. Based on recent
findings in murine models, indicating that the lack of expression of
Jak3 in the periphery is associated with accumulation of activated, anergic CD4+ T cells in the periphery,21 it is
likely that defective immune homeostasis in the periphery accounts for
the development of oligoclonal, activated, and anergic T cells in our
patient.
Accordingly, the data relative to the TCR diversity further support the
hypothesis of an ongoing clonal expansion. The TCRBV repertoire of this
patient was not simply restricted in terms of TCRBV genes usage; each
of the dominant TCRBV families expressed were found to be oligoclonal.
The presence of a limited number of available TCRBV chain molecules may
limit antigenic recognition and thus contribute to immunodeficiency.
Finally, the observation that our patient developed hemolytic anemia
after BMT, when a persistence of autologous T cells was detected, is in
agreement with the observation that deletion of self-reactive T cells
in the thymus and in the periphery is defective in Jak3-deficient
mice,50 and thus adds to the key role played by the
c/Jak3 signaling pathway in T-lymphocyte differentiation and function.
| |
FOOTNOTES |
Submitted April 22, 1997;
accepted September 24, 1997.
Partially supported by grants from Telethon Italy (grant A.42 to
L.D.N.). This paper is manuscript no. 12 of the Genoma 2000/ITBA Project, funded by CARIPLO.
Address reprint requests to Luigi D. Notarangelo, MD, Clinica
Pediatrica, Spedali Civili Brescia, Piazza Spedali Civili 1, 25123 Brescia, Italy.
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.
| |
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Abstract
Introduction
Abstract
