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IMMUNOBIOLOGY
From the Laboratoire de Parasitologie, Faculté de
Médecine and the Département de Génétique
Médicale, Hôpital Erasme, Université Libre de
Bruxelles, Brussels, Belgium; CUMETROP/LABIMED, Faculdad de
Medecina, Universidad Mayor de San Simon, Cochabamba, Bolivia; and
Institut National de la Santé et de la Recherche
Médicale U277, Département d'Immunologie,
Institut Pasteur, Paris, France.
Fetal/neonatal immune responses generally are considered to be
immature and weaker than that of adults. We have studied the cord-blood
T cells of newborns congenitally infected with Trypanosoma cruzi, the protozoan agent of Chagas disease. Our data
demonstrate a predominant activation of CD8 T cells expressing
activation markers and armed to mediate effector functions. The
analysis of the T-cell receptor beta chain variable repertoire
shows the oligoclonal expansion of these T lymphocytes, indicating that activation was driven by parasite antigens. Indeed, we have detected parasite-specific CD8 T cells secreting interferon- The morbidity and mortality rates of infectious
diseases are highest among neonates and young children. This increased
susceptibility has been related to the immaturity of the neonatal
immune system and a bias toward a Th2-type response generally not best
suited to fight intracellular pathogens.1 The majority of
cord-blood T cells present the CD45RA+ naive phenotype and
a decreased capacity to produce interferon- Trypanosoma cruzi, the protozoan agent of Chagas disease, is
transmitted to humans congenitally or by blood-suckling vector insects
or blood transfusions. After a generally mild acute illness, infected
people (16 to 18 million in Latin America) enter a chronic phase, and
10 to 20 years later nearly 30% of them develop cardiac and/or
gastrointestinal lesions, which can lead to death.7 Congenital transmission of T cruzi occurs in 1% to 10% of
chronically infected mothers.8 Since mechanisms implied in
protective immunity in Chagas disease likely involve CD8 T
cells,9 we have addressed the issue of a possible
cord-blood CD8 T-cell response in congenitally T
cruzi-infected neonates. We found that fetuses are able to
generate a massive and oligoclonal expansion of effector/memory CD8 T
cells directed toward a live pathogen.
Patients
Cell sample isolation and cultures
Flow cytometry analysis We performed 3-color flow cytometry analysis of CBMCs' surface by using various combinations of the following monoclonal antibodies (mAbs) and their matched control isotypes: CD3-PercP, CD4-fluorescein isothiocyanate (FITC); CD8-phycoerythrin (PE), CD8-FITC, HLA-DR-FITC; CD69-FITC, CD45RO-PE, V 3, V5 (Becton
Dickinson, Erembodegem, Belgium), V11, V13.1, V14, V16,
V17, V18, V22, T-cell receptor-  , and CD25 (Immunotech,
Marseille, France), all conjugated to PE. We analyzed cells using a
CD3+ cell gate, and we considered CD8 or
CD4 T cells as CD4+ and
CD8+ T lymphocytes, respectively, and
TCR- T cells as TCR- + T cells. We
performed data acquisition and analysis using a Becton Dickinson
fluorescence-activated cell-sorter scanner (FACScan) flow
cytometer and CELLQuest software (Becton Dickinson).
We examined intracellular effector molecules with PE-conjugated mAbs
antihuman IFN- Apoptosis assay Following 24 hours in culture medium supplemented or not with IL-15 (1 ng/mL), we recovered and stained CBMCs with anti-CD8-PE and anti-CD3-Percp. We then determined the extent of spontaneous cell death by staining CBMCs with FITC-conjugated annexin-V (Becton Dickinson). We quantified the level of cell death as the percentage of annexin-V-positive T cells. We also analyzed apoptosis using the Tdt mediated Utp nick end labeling (TUNEL) assay as recommended by the manufacturer (Immunotech).Enzyme-linked immunospot assay for single-cell IFN- mAb (Mabtech, Stockholm, Sweden). We cultured cells
for 24 hours, and we performed the IFN- Elispot (enzyme-linked
immunospot) assay as previously described.13 Spots were
counted with an AID Elispot Reader System (Autoimmun Diagnostika GmbH,
Strasberg, Germany). Responses were considered significant if (1) a
minimum of 5 spot-forming cells (SFCs) were present per well, and (2) this number was at least 2-fold more than obtained with the
negative control.
TCR repertoire analysis We analyzed beta chain CDR3 size distributions using the reverse transcriptase-polymerase chain reaction (RT-PCR)-based Immunoscope technique previously described.14 Briefly, we extracted total RNA from approximately 10 million cells per sample using Trizol (Life Technologies [Gibco BRL], Cergy Pontoise, France). Twenty-five percent of the total RNA was reverse transcribed into cDNA, to be further amplified in 40-cycle PCR reactions using 24 BV-specific primers, each paired with one beta-chain constant region (BC) primer. We then copied the 24 PCR amplification products in a 5-cycle run-off reaction primed with a nested fluorescent BC primer. We loaded aliquots of the labeled BV-BC products on an Applied Biosystems (Foster City, CA) 373 DNA sequencer, and we determined their size distribution with the help of the Immunoscope software.Fluorescence in situ hybridization assay We evaluated the proportion of maternal cells in CBMCs from male newborns by fluorescence in situ hybridization (FISH) assay using centromeric probes for the chromosomes X (CEPX, Xp11.1-q11.1, locus DXZ1, spectrum green) and Y (CEPY, Yp11.1-q11.1, locus DYZ3, spectrum orange) (Vysis, Downers Grove, IL). The preparation of the slides and the FISH analysis have been described in detail elsewhere.15 We counted a total of 300 cells in each experiment using a fluorescent microscope (Zeiss Axioskop, Thornwood, NY) equipped with single-band pass filters (Vysis).
CBMCs of congenitally T cruzi-infected newborns contain a high percentage of activated CD8 T cells We initially observed a marked decrease of the CD4/CD8 mean ratio in congenitally infected newborns compared with uninfected neonates (1.6 ± 0.3, n = 11; vs 3.7 ± 0.3, n = 32; respectively, Student test: P < .01). As expected, analysis of the expression of memory/activation markers indicated T cells from uninfected newborns being naive, since few cells expressed CD45RO and HLA-DR markers (Table 1). By contrast, the percentage of both CD8+CD45RO+ and CD8+HLA-DR+ T cells was strongly and significantly increased in all congenitally infected neonates. We also observed a significant rise in the number of CD4+ T cells expressing CD45RO and HLA-DR, although at a level much lower than that observed within the CD8 T-cell subset. Conversely, we did not observe any difference in the percentage of T cells expressing CD69 or CD25 molecules between both groups. To further characterize the presence of highly differentiated CD8 T cells, we extended our analysis to the expression of the costimulatory CD28 molecule, since CD8+CD28 T cells are defined as end-stage
effectors.16 Our results clearly showed a significant
degree of CD28 loss in congenitally infected newborns, predominantly
within the CD8 T-cell population (Table 1). Taken together, these data
illustrate that congenital transmission of T cruzi is
associated with a strong activation of CD8 T cells.
We next asked if activated T cells in cord blood of congenitally infected newborns might correspond to transferred maternal cells (materno-fetal microchimerism).17 To test this possibility, we used the dual-color FISH assay to measure the proportion of maternal cells within CBMCs from one congenitally infected and one uninfected male newborn. The FISH assay allowed detection of normal rates of materno-fetal microchimerism of 0.3% and 1.6% in cord blood from congenitally infected (containing 61.6% HLA-DR+ and 35.8% CD45RO+ CD8 T cells) and uninfected newborns, respectively. These data indicate that activated T cells in congenitally infected newborns are fetal and not maternally derived lymphocytes. T cells in congenitally infected newborns display restricted TCR
V T cells over TCR- T cells among
CD3+CD45RO+-positive T lymphocytes from
congenitally infected newborns (92.2 ± 1.6%, n = 9; vs
77.3 ± 3.0%, n = 25; respectively, Mann-Whitney test:
P < .05). To investigate if activated cord-blood T cells had defined TCR- repertoires, we analyzed by flow cytometry the
proportions of CD8 and CD4 T cells expressing the TCR-V3, 5, 11, 13.1, 14, 16, 17, 18, and 22 subtypes in 3 congenitally infected
neonates and 6 to 9 control newborns. We have arbitrarily chosen this
panel of TCR V chains that covered around 30% of the CD8 V
repertoire and 25% of the CD4 V repertoire in the control group.
The percentages of CD8 T cells expressing V5, V13.1, and V17
in congenitally infected newborn 1, V13.1 and V22 in newborn 2, and V16 in newborn 3 were increased above the upper
threshold (mean + 2SD) of control newborns (Table
2). We also detected a low expansion of
CD4 T cells expressing V5 and V13.1 in newborn 1, nearly 2 times
less than those obtained within CD8 T cells. Furthermore, it is worth
pointing out that newborns 1 and 2 displayed a low increase in the
percentage of CD4 T cells expressing V11 and/or V18, whereas we
did not detect up-regulation for this V family within the CD8 T-cell
subset. These results demonstrate that mainly the cord-blood CD8 T
cells of congenitally infected newborns display a restricted TCR
V repertoire.
To characterize the clonality of the expanded T-cell populations, we
analyzed CDR3 size patterns of TCR-BV chain transcripts. In agreement
with previous work,18 cord-blood T cells from uninfected newborns displayed a gaussian CDR3 size pattern in all BV families (data not shown), revealing their diverse polyclonal TCR BV
repertoires. By contrast, the analysis of CBMCs from 3 congenitally
infected newborns showed nongaussian profiles, with the presence of
dominant peaks in several BV subfamilies as well as in beta-chain
junction region (BJ) families. Typical TCR BV pattern for
newborn 2 is shown in Figure 1.
Interestingly, we found the peak in BV13A from Figure 1 to correspond
mainly to a BV13A-BJ1S5 rearrangement that could represent as little as
one clone recognized by the BV13.1 antibody used in the flow cytometry
studies (not shown). Thus, while the polyclonal repertoire of healthy
newborns reflects the lack of T-cell priming by previous
antigenic exposure, both the clear clonal T-cell expansion and the
presence of activated and expanded CD8 T cells in congenitally infected
newborns suggest that fetal CD8 T-lymphocyte activation was driven by
parasite antigens.
CD8 and CD4 T cells from congenitally infected newborns are differentiated into potential effector T cells We sought for a relationship between the activated T-cell phenotype and effector functions. For this purpose, we investigated by flow cytometry the ability of both CD8 and CD4 T cells to produce intracellular effector cytokines upon short in vitro restimulation with PMA and ionomycin. The percentage of cord-blood CD8 T cells positive for intracellular IFN- and TNF- was significantly higher in
congenitally infected newborns than in the control group (Table 1). We
also detected a greater mean fluorescence intensity (MFI) for TNF-
expression within CD8 T cells from congenitally infected neonates
(MFI ± SEM, 120.6 ± 22.4 vs 26.1 ± 3.4 in the control group), indicating a better ability to produce this cytokine. The
percentage of CD4 T cells producing IFN- and the MFI of TNF- expression within this cell subset (MFI, 46.3 ± 9.0 vs 29.6 ± 2.6
in the control group) were also significantly, albeit slightly, increased in congenitally infected newborns. In contrast, the proportions of IL-2- or IL-4-producing T cells were similar in both
groups of newborns.
The ability of fetal T cells from infected newborns to exhibit effector cytokines prompted us to measure the intracellular expression of the lytic molecule perforin in unstimulated CBMCs. We observed a significant enrichment in the percentage of both CD8 and CD4 T cells positive for perforin in congenitally infected newborns as compared with the control group (Table 1). Altogether, these results show that activated T cells of congenitally infected newborns are armed to exert effector functions. Congenitally T cruzi-infected newborns suffer a high level of CD8 T-cell spontaneous apoptosis Generally, the overwhelming numbers of activated CD8 T cells generated following infection are associated with T-cell death.19 We therefore evaluated the level of spontaneous T-cell death using annexin-V staining after CBMC culture for 24 hours in the presence or absence of IL-15 known to improve T-cell survival20 (Figure 2). CD8 T cells from congenitally infected newborns underwent a marked level of spontaneous cell death (57.4 ± 4.0%) as compared with uninfected neonates (19.4 ± 3.0%). We also found a significant and higher cell death level within the CD4 T-cell subset (22.2 ± 2.1% vs 13.7 ± 1.9% in control group). The TUNEL assay applied on CBMCs from 2 patients confirmed that T cells from congenitally infected newborns were prone to die by apoptosis (Figure 2C-D). Moreover, addition of IL-15 (Figure 2B) to the cell culture prolonged the survival of CD8 T cells, which is consistent with a mitochondrial/cytokine rescuable pathway of apoptotic cell death.20
Cord blood from congenitally infected newborns contains parasite-specific CD8 T cells Given the vigorous CD8 T-cell response in congenitally infected newborns, we analyzed the presence of parasite-specific cord-blood T cells using the ex vivo IFN- Elispot assay (Figure
3). We used live parasites to further
enhance CD8 T-cell stimulation through a major histocompatibility
complex (MHC) class I presentation. CBMCs were costimulated or not with
IL-15 since this cytokine is known to: (1) improve T-cell responses in
intracellular infection,21 (2) prevent apoptosis (see
above), and (3) enhance the division of CD8+ memory
pool.22 We observed IFN--secreting cells from 7 of 9 (77.8%) congenitally infected newborns when IL-15 was added, while 3 of 12 (25%) displayed positive responses following stimulation with
live T cruzi alone (Figure 3A). These responses could not be
related to an unspecific production of IFN-, since cells from uninfected newborns failed to produce this cytokine, even after addition of IL-15. Likewise, flow cytometry analysis of CBMCs exposed
to both IL-15 and live parasites showed that CD8 T cells from
congenitally infected newborns expressed detectable amount of
intracellular IFN- in 2 of 3 donors tested (Figure 3B-E). These
results provide evidence that parasite-specific CD8 T cells are present
in cord blood from congenitally infected newborns.
The present work shows for the first time a massive and oligoclonal development of fetal CD8 T cells following congenital infection with an intracellular pathogen. These cells display a phenotype of activated effector T cells, and a substantial number of them are parasite specific. This in utero immune response of congenitally T cruzi-infected newborns mimics the strong antigen-specific CD8 T-cell expansion previously described in adults during acute viral infections.23,24 Furthermore, while a CD8+ T-cell-mediated response contributes to the control of acute parasitemia in experimental Chagas disease,25 no data are currently available concerning the role of CD8 T cells during acute human infection with T cruzi. Increased T-cell apoptosis is a classical feature of T-cell activation
following immune response in vivo.19 In our study, IL-15
rescued activated T cells from spontaneous apoptosis and improved the
ability of parasite-specific CD8 T cells to produce IFN- The tremendous expansion of CD8 T cells was associated with major
changes in surface phenotypes, which are closely related to acquired
effector functions. Along this line, the expression of effector
molecules such as IFN- Although we observed a preponderant CD8 T-cell response, we also
detected low levels of activated CD4 T cells in cord blood from
congenitally infected newborns, and the analysis of V However, our results do not support a previous report about a
preferential modulation of V To date, the correlation between the observed CD8 T cells in T cruzi congenitally infected newborns and an immunoprotective role later in life remains to be determined. However, our data support the idea that the fetal immune system is more competent than previously appreciated. The demonstration that maturation of a CD8 T-cell immune response may occur in utero looks promising in the field of neonatal vaccination to control early infections with intracellular pathogens.
We thank Eduardo Suarez and Marisol Cordova and the staff of the maternity German Urquidi (Cochabamba, Bolivia) for the management of patients; Rudy Parrado and Myriam Huanca (Centro Universitario de Medicina Tropicale/Laboratorio de Biologica Medical [CUMETROP/LABIMED], Universidad Mayor de San Simón, Cochabamba, Bolivia) for T cruzi diagnosis of patients; and Samira Benyoucef Dahmani (Cytokine Profile [CYPRO SA]) for helping with the Elispot analysis. We are grateful to Michel Goldman and Eric Muraille for their comments on the manuscript.
Submitted August 21, 2001; accepted May 8, 2002.
Supported by the Centre de Recherche Interuniversitaire en Vaccinologie (CRIV) and sponsored by the Région Wallonne and Glaxo-Smithkline biologicals (Belgium) and by the Conseil Interuniversitaire de la Communauté française de Belgique (CIUF).
E.H. and C.A.-V. are research fellows of CRIV and Association pour la promotion de l'éducation et la formation à l'étranger (APEFE, Communauté Française de Belgique), respectively.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Yves Carlier, Laboratoire de Parasitologie, Faculté de Médecine, U.L.B. Route de Lennick, 808, CP 616, B-1070 Brussels, Belgium; e-mail: ycarlier{at}ulb.ac.be.
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F. TORRICO, C. ALONSO-VEGA, E. SUAREZ, P. RODRIGUEZ, M.-C. TORRICO, M. DRAMAIX, C. TRUYENS, and Y. CARLIER MATERNAL TRYPANOSOMA CRUZI INFECTION, PREGNANCY OUTCOME, MORBIDITY, AND MORTALITY OF CONGENITALLY INFECTED AND NON-INFECTED NEWBORNS IN BOLIVIA Am J Trop Med Hyg, February 1, 2004; 70(2): 201 - 209. [Abstract] [Full Text] [PDF]
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M. Salio, N. Dulphy, J. Renneson, M. Herbert, A. McMichael, A. Marchant, and V. Cerundolo Efficient priming of antigen-specific cytotoxic T lymphocytes by human cord blood dendritic cells Int. Immunol., October 1, 2003; 15(10): 1265 - 1273. [Abstract] [Full Text] [PDF]
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| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
, n = 17) and congenitally
infected (
, n = 12) newborns. **P < .01 vs
uninfected newborns (Student test). (B) Mean percentage ± SEM of
spontaneous T-cell death following cell culture for one day in the
presence or absence of IL-15. (C, D) Two representative experiments, in
one uninfected (C) and one congenitally infected neonate (D) of the
TUNEL assay analysis of spontaneous T-cell death are shown. Percentages
of cellular spontaneous apoptosis are indicated in bold.
#P < .05 vs unstimulated CBMCs (Student test).
