Human cytomegalovirus elicits fetal gammadelta T cell responses in utero.

Abstract

The fetus and infant are highly susceptible to viral infections. Several viruses, including human cytomegalovirus (CMV), cause more severe disease in early life compared with later life. It is generally accepted that this is a result of the immaturity of the immune system. gammadelta T cells are unconventional T cells that can react rapidly upon activation and show major histocompatibility complex-unrestricted activity. We show that upon CMV infection in utero, fetal gammadelta T cells expand and become differentiated. The expansion was restricted to Vgamma9-negative gammadelta T cells, irrespective of their Vdelta chain expression. Differentiated gammadelta T cells expressed high levels of IFN-gamma, transcription factors T-bet and eomes, natural killer receptors, and cytotoxic mediators. CMV infection induced a striking enrichment of a public Vgamma8Vdelta1-TCR, containing the germline-encoded complementary-determining-region-3 (CDR3) delta1-CALGELGDDKLIF/CDR3gamma8-CATWDTTGWFKIF. Public Vgamma8Vdelta1-TCR-expressing cell clones produced IFN-gamma upon coincubation with CMV-infected target cells in a TCR/CD3-dependent manner and showed antiviral activity. Differentiated gammadelta T cells and public Vgamma8Vdelta1-TCR were detected as early as after 21 wk of gestation. Our results indicate that functional fetal gammadelta T cell responses can be generated during development in utero and suggest that this T cell subset could participate in antiviral defense in early life.

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J. Exp. Med. Vol. 207 No. 4 807-821
www.jem.org/cgi/doi/10.1084/jem.20090348
807
The fetus and young infant have a high suscep-
tibility to infections with intracellular patho-
gens, suggesting that T cell–mediated immune
responses are different in early life. A number
of viruses, including human CMV, herpes sim-
plex type 2, respiratory syncytial virus, and
HIV, cause more severe or rapidly progressive
disease in early life as compared with later life
(Stagno, 2001; Marchant and Goldman, 2005).
It is generally accepted that this increased sus-
ceptibility to viral infections is related to the
immaturity of the neonatal immune system.
This includes intrinsic defects of conventional
T cells, especially CD4  T cells, and im-
paired DC responses (Lewis and Wilson, 2001;
White et al., 2002; Maródi, 2006; Levy, 2007;
Lee et al., 2008). CMV is the most common
cause of congenital infection, affecting 0.2% of
all live births in industrialized countries and up
to 3% in developing countries (Stagno, 2001).
Although CMV infection causes no detectable
symptoms in immunocompetent adults, 20%
of newborns with congenital infection develop
serious symptoms, including cerebral malforma-
tions, multiple organ failure, deafness, and mental
retardation (Stagno, 2001; Dollard et al., 2007).
 T cells are T cells expressing  and 
chains as a TCR on their cell surface instead of
 and  chains as in conventional CD4 and
CD8  T cells. Together with  T cells,
they have been conserved for >450 million
years of evolution (Hayday, 2000).  T cells
are the prototype of unconventional T cells;
they can react rapidly upon activation and
show MHC-unrestricted activity (Hayday, 2000;
Holtmeier and Kabelitz, 2005). Thus, they are
not influenced by MHC down-regulation strat-
egies used by viruses such as CMV to escape
conventional T cells (Wilkinson et al., 2008).
CORRESPONDENCE
David Vermijlen:
dvermijl@ulb.ac.be
Abbreviations used: CDR3,
complementary-determining-
region-3; KIR, killer immuno-
globulin receptor; MFI, mean
fluorescence intensity; NKR,
NK receptor.
Human cytomegalovirus elicits fetal
 T cell responses in utero
David Vermijlen,1 Margreet Brouwer,1 Catherine Donner,2 Corinne Liesnard,3
Marie Tackoen,4 Michel Van Rysselberge,5 Nicolas Twité,1 Michel Goldman,1
Arnaud Marchant,1 and Fabienne Willems1
1Institute for Medical Immunology, Université Libre de Bruxelles, 6041 Gosselies, Belgium
2Department of Obstetrics and Gynecology and 3Department of Virology, Hôpital Erasme, 1070 Brussels, Belgium
4Neonatal Intensive Care Unit and 5 Fetal Medicine Unit, Department of Obstetrics and Gynecology, Le Centre Hospitalier
Universitaire Saint-Pierre, 1000 Brussels, Belgium
The fetus and infant are highly susceptible to viral infections. Several viruses, including
human cytomegalovirus (CMV), cause more severe disease in early life compared with later
life. It is generally accepted that this is a result of the immaturity of the immune system.
 T cells are unconventional T cells that can react rapidly upon activation and show major
histocompatibility complex–unrestricted activity. We show that upon CMV infection in
utero, fetal  T cells expand and become differentiated. The expansion was restricted to
V9-negative  T cells, irrespective of their V chain expression. Differentiated  T cells
expressed high levels of IFN-, transcription factors T-bet and eomes, natural killer
receptors, and cytotoxic mediators. CMV infection induced a striking enrichment of a
public V8V1-TCR, containing the germline-encoded complementary-determining-
region-3 (CDR3) 1–CALGELGDDKLIF/CDR38–CATWDTTGWFKIF. Public V8V1-TCR–
expressing cell clones produced IFN- upon coincubation with CMV-infected target cells
in a TCR/CD3-dependent manner and showed antiviral activity. Differentiated  T cells
and public V8V1-TCR were detected as early as after 21 wk of gestation. Our results
indicate that functional fetal  T cell responses can be generated during development in
utero and suggest that this T cell subset could participate in antiviral defense in early life.
© 2010 Vermijlen et al. This article is distributed under the terms of an Attribu-
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The Journal of Experimental Medicine

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Antiviral  T cells in utero | Vermijlen et al.
correlated with a higher percentage of  T cells express-
ing the proliferation marker Ki-67 in CMV-infected new-
borns (Fig. 1 C).
The expansion of  T cells in CMV-infected newborns
is restricted to V9 cells, irrespective of the usage
of the V chain
To further define specific subsets of  T cells in cord blood
of CMV-infected newborns, flow cytometry analysis was per-
formed with antibodies specific against V9, V1, V2, and
V3. In combination with the pan- TCR antibody, the
V9 antibody can make distinction between V9+ and V9
Studies in several species have shown an important role for 
T cells in protection against infection, in tumor surveillance, in
immunoregulation, and in tissue repair (Hayday, 2000; Wang
et al., 2001; Holtmeier and Kabelitz, 2005; Pennington et al.,
2005; Toulon et al., 2009). In general, they show a rapid and
robust response before the development of the adaptive im-
munity mediated by conventional T cells. In comparison with
 T cells,  T cells are not abundant in the peripheral blood
but are highly enriched in tissues like the gut epithelium
(Hayday, 2000; Holtmeier and Kabelitz, 2005). The majority
of  T cells in human adult peripheral blood use the TCR
V region pair V9V2 (note that according to an alternative
nomenclature the V9 chain is also termed V2 [Holtmeier
and Kabelitz, 2005]). This subset has been shown to react
specifically toward nonpeptide low molecular weight phos-
phorylated metabolites (so-called phosphoantigens) and has
been the subject of several clinical trials (Wilhelm et al., 2003;
Dieli et al., 2007; Kabelitz et al., 2007).
Probably in all species,  T cells are the first T cells to
develop (Hayday, 2000). In contrast to adult peripheral blood
 T cells, human neonatal cord blood  T cells express di-
verse V and V chains paired in a variety of combinations
(Morita et al., 1994). Thus the adult-like V9V2 subpopu-
lation only represents a small fraction of the neonatal 
T cells (Parker et al., 1990; Morita et al., 1994; Cairo et al.,
2008). Further illustrating the differences between adult and
neonatal  T cells, is the demonstration that in vitro expo-
sure toward the same pathogen (Escherichia coli or Pseudomonas
aeruginosa) results in expansion of V2+  T cells in adult
peripheral blood but of V1+  T cells in cord blood (Kersten
et al., 1996). In mice,  T cells are important for the protec-
tion against an intestinal parasite infection in early life but not
in adult life (Ramsburg et al., 2003), and during human T cell
ontogeny  T cells mature before  T cells (De Rosa et al.,
2004). However, so far it is not known whether pathogens in
early life can activate human  T cells. To gain insight into
the ability of  T cells to mount responses to viruses during
fetal life, we studied the changes occurring in the  T cell
compartment during congenital CMV infection.
RESULTS
CMV infection in utero induces expansion of fetal
 T cells in newborns
To address whether human fetal  T cells are responsive to
CMV infection in utero, we first compared the percentage of
 T cells among all T cells in cord blood samples derived
from 19 CMV-infected newborns versus 22 control CMV-
uninfected newborns. In CMV-infected newborns, the
percentage of  T cells was significantly higher than in
CMV-uninfected newborns (Fig. 1 A). To exclude the pos-
sibility that this higher percentage of  T cells was the result
of a decreased number of  T cells, we determined the
absolute number of  T cells per microliter of blood. In-
deed, significantly more  T cells were present per micro-
liter of cord blood in CMV-infected newborns in comparison
with controls (Fig. 1 B). The higher number of  T cells
Figure 1. CMV infection in utero induces an expansion of 
T cells in newborns. (A) Percentage of  T cells of total T cells (CMV+,
n = 19; CMV, n = 22). (B) Absolute number of  T cells per microliter of
cord blood (CMV+, n = 13; CMV, n = 15). (C) Percentage of  T cells
which are Ki-67+ (CMV+, n = 9; CMV, n = 15) in CMV-infected (gray
boxes) and CMV-uninfected (white boxes) newborns. In box-and-whisker
graphs, the line at the middle is the median, the box extends from the
25th to 75th percentile, and the error bars, or whiskers, extend down to
the lowest value and up to the highest.

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809
cells negative for V9, including V9V1+, V9V2+, and
V9V3+  T cells in CMV-infected newborns compared
with uninfected newborns (Fig. 2, A and B). On a selected
number of CMV-uninfected and CMV-infected newborns,
we performed a more detailed analysis of the  chain usage.
In CMV-uninfected newborns, there was a slight preference for
V4 and V9, whereas upon CMV infection the VI family
members V4 and V8 were highly expanded (Fig. S1).
 T cells (Fig. 2 A). V9 is the only member of the VII
family; thus the V9 cells express V chains of the VI fam-
ily (Hayday, 2000). The combination of V1, V2, and V3
antibodies stained the vast majority (90%; unpublished
data) of the cord blood  T cells. This approach allows us
to identify six  T cell subpopulations in cord blood:
V9+V1+, V9V1+, V9+V2+, V9V2+, V9+V3+,
and V9V3+. We detected higher percentages of  T
Figure 2. The expansion of  T cells in CMV-infected newborns is restricted to V9  T cells, irrespective of the usage of the
V chain. (A) Expression of V9 versus V1, V2, or V3 by  T cells from CMV-infected newborn Pos13 (top) and CMV-uninfected newborn Neg4 (bottom).
Numbers in dot plots are the percentages of total T cells. (B) Box-and-whisker graph (defined as in Fig. 1) of the percentages of V9+V1+, V9+V2+,
V9+V3+, V9V1+, V9V2+, and V9V3+  T cell subpopulations of  T cells expressed as a percentage of total T cells. CMV+, n = 10–18;
CMV, n = 14–22.

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Antiviral  T cells in utero | Vermijlen et al.
NKRs. Expression of a range of NKR genes was increased in
 T cells from CMV-infected newborns in comparison with
 T cells from CMV-uninfected newborns (Fig. 3, KIR2DL1;
Table S1). This included both activating and inhibitory re-
ceptors (Table S1) and involved all the NKR families: the
killer immunoglobulin receptor (KIR) family, the C-type lec-
tin family (CD94/NKG2A/NKG2C), and the natural cyto-
toxicity receptor (NCR) family (NKp46; Lanier, 2008). In
CMV-uninfected newborns, there were either no or very few
 T cells (CD94/NKG2A/NKG2C, CD158a/h [KIR2DL1/
KIR2DS1], and CD158b/j [KIR2DL2/KIR2DS2]) or a
significant fraction of  T cells (NKG2D and KLRG1) ex-
pressing NKR on their membrane, as determined by flow
cytometry (Table I). In CMV-infected newborns, signifi-
cantly more  T cells expressed all these NKRs (Table I).
Cytotoxic mediators. In general, cytotoxic lymphocytes can
kill target cells by two main mechanisms: exocytosis of granule-
associated molecules, such as granzymes, perforin, and gra-
nulysin, or binding to receptors with ligands of the TNF
superfamily (e.g., FasL and TRAIL). Among the >47,000
transcripts analyzed, the two genes displaying the most in-
creased expression upon CMV infection were members of the
granzyme family: granzyme B and granzyme H (Fig. 3). In
addition, other granzyme family members (granzyme A and
granzyme M), perforin, granulysin, FasL, and TRAIL were
increased (Table S1). In CMV-uninfected newborns, there
was either no or only a low percentage of  T cells express-
ing perforin and granzyme A, as demonstrated by flow cytom-
etry (Table I). In CMV-infected newborns, the percentages of
 T cells expressing perforin or granzyme A were highly
increased (Table I). This expression was clearly associated with
the late differentiation status of the  T cells (Fig. S2 A).
Chemokines and chemokine receptors. The genes for the
chemokines CCL3 (MIP-1), CCL4 (MIP-1), and CCL5
 T cells from CMV-infected newborns are activated
and differentiated
Next, we evaluated whether the expansion of fetal  T cells
after congenital CMV infection was accompanied by activa-
tion and/or differentiation of these cells. A significant pro-
portion of  T cells from CMV-infected newborns expressed
the activation marker HLA-DR, whereas expression was
virtually absent in uninfected controls (Table I). Down-
regulation of CD27 and CD28 expression has been shown to
be associated with advanced or late differentiation in CD8
 T cells upon CMV infection (Appay et al., 2002; Marchant
et al., 2003; van Leeuwen et al., 2006). These markers
have also been used to identify differentiated human 
T cells (Morita et al., 2007). Although CD27CD28 
T cells were absent from CMV-uninfected newborns, a large
proportion of  T cells showed this phenotype in CMV-
infected newborns (Table I). This differentiation was most
pronounced in the V9  T cell subpopulation (unpub-
lished data). Collectively, these data clearly show that
upon congenital CMV infection,  T cells are activated,
undergo cell division, and become differentiated.
Expression of NK receptors (NKRs), cytotoxic mediators,
and IFN- is highly increased in  T cells
of CMV-infected newborns
To gain insight into the function of fetal  T cells in new-
borns with congenital CMV infection, we compared the
gene expression profiles of  T cells derived from three
CMV-infected newborns versus three CMV-uninfected new-
borns. 1,622 genes were increased and 654 decreased upon
infection (using the selection criteria described in Materials
and methods; M > 0.05, P < 0.05). More than 100 genes as-
sociated with cell cycle showed increased expression upon
CMV infection (as analyzed with DAVID; not depicted),
coinciding with the expansion data (Fig. 1).
Table I. Percentage of activation (HLA-DR) and differentiation (CD27, CD28) markers, NKRs (CD94, NKG2A, NKG2C, CD158,
NKG2D, and KLRG1), cytotoxic mediators (perforin and granzyme A), and chemokine receptor CX3CR1 on  T cells derived from
CMV-infected and CMV-uninfected newborns
Marker CMV+
CMV
p (CMV+ versus CMV)
HLA-DR+
CD27CD28
CD94+
NKG2A+
NKG2C+
CD158a/h+
CD158b/j+
NKG2D+
KLRG1+
perforin+
granzyme A+
CX3CR1+
6.34 (2.87–10.68)
43.82 (31.97–48.98)
33.21 (23.68–57.20)
10.99 (4.27–20.64)
28.00 (10.77–35.54)
14.70 (6.61–30.53)
46.60 (29.07–51.38)
69.18 (64.42–76.76)
38.85 (27.80–45.41)
61.50 (51.97–73.32)
69.82 (57.97–79.63)
43.21 (25.08–57.96)
0.58 (0.27-0.80)
0.22 (0.13-0.45)
4.44 (3.61-5.56)
3.84 (2.33-5.11)
1.17 (0.91–3.68)
1.56 (0.94–3.34)
2.75 (2.56–3.32)
52.38 (46.26–63.08)
24.70 (16.73–28.72)
0.67 (0.48–1.77)
4.38 (2.82–6.28)
1.11 (0.65–2.10)
<0.0001
<0.0001
<0.0001
0.0464
<0.0001
<0.0001
<0.0001
0.0001
0.0009
<0.0001
<0.0001
0.0003
Data for CMV+ and CMV represent median (25–75% percentile). The gate is put on CD3++. CMV+, n = 8–18; CMV, n = 9–22.

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measured by the mean fluorescence intensity (MFI), was
consistently much higher in CD27CD28  T cells than
in CD27+CD28+  T cells (median within CD27CD28
 T cells, 318 MFI; median within CD27+CD28+ 
T cells, 102 MFI; P = 0.0023). Upon a brief polyclonal stim-
ulation in vitro, the majority of CD27CD28 differentiated
 T cells of CMV-infected newborns produced IFN-
(Fig. 4 B), whereas the CD27+CD28+  T cells produced
significantly less IFN- (median within CD27CD28 
T cells, 68%; median within CD27+CD28+  T cells, 22%;
P = 0.0006). T-bet and IFN- expression within  T cells
from CMV-uninfected newborns were similar to the expres-
sion found within CD27+CD28+  T cells from CMV-
infected newborns (unpublished data).
The CDR31 and CDR32 are highly restricted
upon congenital CMV infection
To study the impact of CMV infection during fetal life on
the TCR repertoire of  T cells, we assessed the degree of
junctional diversity of the complementary-determining-
region-3 (CDR3) of the V1 (CDR31) and V2 (CDR32)
chains by spectratyping on 11 CMV-uninfected and 13
CMV-infected cord blood samples. CMV-uninfected cord
blood samples showed polyclonal profiles for both CDR31
(as described previously; Beldjord et al., 1993) and CDR32.
In contrast, the CDR31 and CDR32 repertoires became
highly restricted in the vast majority of CMV-infected
newborns (Fig. 5 A and Fig. S3). To quantify this restriction,
we calculated an index of oligoclonality for CDR31 and
CDR32 as described previously (Déchanet et al., 1999; Pitard
et al., 2008). For both CDR3s, the index of oligoclonality
(RANTES), all ligands for CCR5, and two chemokine re-
ceptor genes (CCR5 and CX3CR1) showed increased
expression in  T cells from CMV-infected newborns in
comparison with  T cells from CMV-uninfected newborns
(Table S1). CCR7 gene expression was decreased upon
CMV infection (M = 2.27, A = 8.07, P = 0.03). In CMV-
uninfected newborns, there were no or only a low percent-
age of  T cells expressing CX3CR1 (fractalkine receptor)
on their membrane, as determined by flow cytometry (Table I).
In CMV-infected newborns, the percentage of  T cells
expressing CX3CR1 was highly increased (Table I), which
was clearly associated with the late differentiation phenotype
of the  T cells (Fig. S2 B).
Cytokines. Only a limited number of cytokine genes showed
increased expression in  T cells derived from CMV-
infected newborns in comparison with  T cells derived
from CMV-uninfected newborns (Table S1). IFN- was one
of the most increased expressed genes (Fig. 3 and Table S1).
Strikingly, gene expression of transcription factors known to
be implicated in IFN- production, namely T-bet (M = 2.37,
A = 6.64, P = 0.000112) and eomes (M = 3.02, A = 6.31,
P = 0.000246), was highly increased. The high expression of
T-bet was confirmed at protein level by flow cytometry in
 T cells from CMV-infected newborns and was associated
with the differentiation status of  T cells (Fig. 4 A).
Almost all CD27CD28  T cells expressed T-bet, whereas
CD27+CD28+  T cells expressed significantly lower levels
of this transcription factor (median within CD27CD28 
T cells, 97%; median within CD27+CD28+  T cells, 28%;
P = 0.0006). In addition, T-bet expression per cell, as
Figure 3. Gene expression analysis of  T cells derived from three CMV-infected newborns versus  T cells derived from three
CMV-uninfected newborns. MA plot of differentially expressed genes in  T cells upon CMV infection. M (log2 of fold change) reflects the differential
expression of a gene. Positive and negative values indicate genes which are up- and down-regulated, respectively, upon CMV infection. A (mean expres-
sion) reflects the overall expression level of a gene. Each dot represents one gene. Examples of highly expressed NKR (KIR2DL1), cytotoxic mediators
(granzyme B, granzyme H, perforin, and granulysin), cytokines (IFN-), and chemokines (CCL4) are indicated.

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Antiviral  T cells in utero | Vermijlen et al.
the c of the joining gene 1 (J1) or completely formed by
the gat of the D3 (Table S2, dark gray). Besides ELGDD
itself, few longer variants were present in some CMV-infected
newborns, which were enriched as well, containing one (Pos3)
or two (Pos3 and Pos11) extra Ts after the first D of ELGDD
(Table S2). In contrast to the other CDR31 sequences, the
highly enriched ELGDD sequence did not contain P/N addi-
tions and was thus completely germline encoded (Table S2).
In comparison with CDR31, the degree of shared
CDR32 sequences among the CMV-infected newborns
was much less clear (Table S3). Among eight CMV-infected
newborns, three exhibited enrichment of the same CDR32
sequence (Pos8, Pos9, and Pos10; Table S3). No other
obvious similarities were found between different enriched
CDR32 sequences of different CMV-infected newborns.
V1 almost always paired with J1, whereas V2 had a pref-
erence for J3 (Table S2 and Table S3). Sequencing of
CDR31 and CDR32 of CMV-uninfected newborns con-
firmed the polyclonal repertoire as found by spectratyping
(Table S2 and Table S3).
We wondered whether the V1 chain, containing the
public CDR31, from CMV-infected newborns cells had a
preference for pairing with specific V chains. By costaining
of V1 and V2/3/4, V5/3, V8, or V9, we determined
that within CMV-uninfected newborns V1 had a preference
was significantly higher in CMV-infected newborns than in
CMV-uninfected newborns (Fig. 5 B). Moreover, the re-
striction of CDR31 of CMV-infected newborns was
enriched for the same length (11 aa) in all CMV-positive
newborns (Fig. 5 A, arrows). This length was either absent or
minimally present in CMV-uninfected newborns (Fig. 5 A).
In contrast to CDR31, the length of the enriched CDR32
sequences in CMV-infected newborns varied from newborn
to newborn (Fig. S3). It is of note that CMV-infected new-
born Pos10 showed a polyclonal CDR31 repertoire corre-
sponding with the absence of CD27CD28 differentiated
V1+  T cells (Fig. 5 A). In contrast, V2+  T cells of this
newborn were well differentiated, corresponding to a re-
stricted CDR32 repertoire (Fig. S3).
Public germline-encoded CDR31 and CDR38 sequences
are highly enriched in CMV-infected newborns
Because the CDR31 of CMV-infected newborns was highly
enriched at 11 aa, we wondered whether this region included
the same or similar sequences. Strikingly, at amino acid level
in all 12 sequenced CMV-infected newborns the CDR31
of 11 aa had exactly the same sequence: CALGELGDDKLIF,
or ELGDD for short (Table S2; Fig. 5 A). At the nucleotide
level, two variants were observed: the first D of ELGDD was
either formed by the ga of the diversity gene 3 (D3) and
Figure 4. Differentiated (CD27CD28)  T cells from CMV-infected newborns express highly the transcription factor T-bet and produce
high levels of IFN-. Flow cytometry plots for T-bet (A) and IFN- (B), gated on CD27CD28 versus gated on CD27+CD28+  T cells of a CMV-positive
newborn (Pos12), representative of seven CMV-infected newborns. Cells were stimulated for 4 h with PMA/ionomycin before intracellular staining for
IFN-. Unstimulated cells showed no IFN- staining.

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Figure 5. The CDR31 and CDR32 repertoire of  T cells from CMV-infected newborns are oligoclonal, and CDR31 is highly enriched
for a single sequence. (A) Spectratyping plots of the CDR31 of CMV-uninfected and CMV-infected newborns. Each box represents one donor. The num-
bers at the left top of each box represents the percentage of V1+  T cells, expressed as percentage of total T cells. The numbers at the right top of each
box represent the percentage of CD27CD28 cells of V1+  T cells. The arrows indicate the sequences at CDR31 of 11-aa size of the CMV-infected
newborns that have been sequenced: CALGELGDDKLIF (Table S2). (B) Index of oligoclonality for CDR31 and CDR32, determined as described in Materials
and methods. Lines indicate medians.

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Antiviral  T cells in utero | Vermijlen et al.
extra Y after D (Fig. 6 B; Table S4). This sequence was not
detected in CMV-uninfected newborns (Table S4). As for the
public CDR31 sequence, the public CDR38 was com-
pletely germline encoded (Table S4).
The public V8V1 TCR reacts against CMV-infected
target cells
To verify whether the public V8V1 TCR reacts against
CMV-infected target cells, we generated  T cell clones ex-
pressing the public TCR containing the CDR31-ELGDD
and CDR38-DTTGW from CMV-infected newborns
Pos4 (11 public clones) and Pos6 (21 public clones). All
clones expressing CDR31-ELGDD coexpressed CDR38-
DTTGW, whereas clones with a different CDR31 expressed
other CDR3’s (unpublished data), showing in a direct way
the preferential pairing between CDR31-ELGDD and
CDR38-DTTGW. A brief coincubation (6 h) of public
for pairing with V2/3/4 (Fig. 6 A), whereas V2 and V3
had rather a preference for V9 and V5/3, respectively (not
depicted). In contrast, in CMV-infected newborns with
highly expanded  T cells, V1 had a clear preference for
pairing with V8 (Fig. 6 A), whereas V2 and V3 had rather
a preference for V2/3/4 (not depicted). Because of this
preferential pairing of V1 with V8, we performed spec-
tratyping for CDR38 on six CMV-uninfected and six
CMV-infected newborns. The CDR38 of CMV-uninfected
newborns showed a polyclonal repertoire. In contrast, the
CDR38 repertoire became highly restricted in CMV-
infected newborns, showing a high enrichment at a length of
11 aa in five out of six CMV-infected newborns. The sixth
CMV-infected newborn (Pos13) had a high enrichment at
12 aa (Fig. 6 B). Sequencing revealed that the CDR38
sequences at 11 aa contained all the same sequence:
CATWDTTGWFKIF (DTTGW for short). Pos13 had an
Figure 6. The V1 chain on  T cells of CMV-infected newborns preferentially pairs with a public V8 chain. (A) The percentage of V1+
 T cells positive for V2/3/4, V5/3, V8, or V9 determined in three CMV-infected (Pos4, Pos6, and Pos13) and three CMV-uninfected newborns.
(B) Spectratyping for CDR38 of six CMV-uninfected newborns (top row) and six CMV-infected newborns (bottom row). The arrow indicates the public
CDR38 sequence CATWDTTGWFKIF of 11 aa (Table S3). The CDR38 of Pos13 contains 1 aa more (Y).

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negative and 13 CMV-positive fetuses). At these earlier
gestation times, the  T cells were already clearly differenti-
ated (down-regulation of CD27 and CD28) and showed
high expression of perforin (Fig. 9 A), granzyme A, and NKR
(not depicted). From four CMV-infected fetuses, we per-
formed spectratyping and sequencing for CDR31 at time of
delivery and at earlier gestation time (Fig. 9 B). We found
that the CDR31-ELGDD sequence was already enriched at
as early as 21 wk of gestation (Fig. 9 B and Table S2). Few
other CDR31 sequences that were present at early gestation
time were also present at time of delivery (Table S2, fetus
Pos4 [14 aa] and fetus Pos13 [17 aa]). As observed at time of
clones with CMV-infected human embryonic lung fibro-
blasts induced IFN- production, which was blocked by the
presence of a soluble anti-CD3 antibody (OKT3) showing
the involvement of the public V8V1 TCR/CD3 complex
in the recognition of CMV-infected target cells (Fig. 7
and Fig. S4). Control  T cell clones of CMV-uninfected
newborns did not show CMV-induced IFN- production.
To gain insight into the antiviral activity of the  T cell
clones, we conducted additional experiments. Public clones
killed infected target cells (Fig. 8 A) and inhibited CMV rep-
lication (between one and two log10 inhibition; Fig. 8 B),
whereas control V9V2 T clones had no or only a moderate
effect (Fig. 8).
Differentiation and oligoclonal expansion of fetal  T cells
can occur early during gestation
To explore the possibility that  T cells could develop a
response toward CMV infection early during fetal life, we
analyzed the  T cells from fetal cord blood samples col-
lected between 20 and 29 wk of gestation (from 12 CMV-
Figure 7.  T cell clones expressing the public V8V1 TCR
display reactivity against CMV-infected cells via TCR/CD3. Clones
were coincubated for 6 h with human embryonic fibroblasts not in-
fected (white bars) or infected (gray bars) with CMV (TB40/E). During
coincubation either a control IgG2a antibody (ctrl) or the anti-CD3 anti-
body OKT3 (anti-CD3) was present in soluble form. Results are shown
for one public clone from CMV-infected newborn Pos4 (V8V1-Pos4)
and for one public clone of CMV-infected newborn Pos6 (V8V1-Pos6)
and are representative of five independent experiments involving 11
different public V8V1 clones.
Figure 8. Public V8V1 clones kill CMV-infected target cells and
inhibit CMV replication in vitro. (A) CMV-infected (TB40/E) human
embryonic fibroblasts were coincubated with either  T cell clones
expressing the public V8V1 TCR (derived from CMV-infected newborns
Pos4 and Pos6) or a control V9V2 clone (derived from a CMV-
uninfected newborn) at the indicated effector to target ratios. After 4 h of
coincubation, the level of DNA fragmentation in the target cells was
quantified. Results are representative of three independent experiments.
(B) Human embryonic fibroblasts were incubated with CMV for 2 h,
washed, and incubated with medium alone, with a public V8V1 clone
from CMV-infected newborn Pos6 or with a control V9V2 clone from a
CMV-uninfected newborn. After 7 d, the quantity of infectious CMV from
the supernatant was determined by a plaque assay (PFU, plaque forming
units). Shown are the mean ± SEM of quadruplicate determinations.
Results are representative of two independent experiments.

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Antiviral  T cells in utero | Vermijlen et al.
infected fetuses and remained present with time. In contrast,
the enriched sequences of the CDR32 were variable from
one fetus to the other and changed during gestation time.
Furthermore, the CDR3 sequence associated with the
CDR31-ELGDD sequence, namely CDR38-DTTGW,
was also already enriched at as early as 21 wk of gestation
delivery, CDR32 spectratyping showed more variability
between fetuses (unpublished data). Furthermore, CDR32
appeared to vary with time within the same fetus (Table S3,
compare CDR32 sequencing data of Pos4 at 20 wk, 5 d and
at 40 wk, 0 d of gestation). Thus, the enriched CDR31-
ELGDD sequence appeared early during gestation in CMV-
Figure 9. Differentiation and oligoclonal (CALGELGDDKLIF) expansion of fetal  T cells can occur early during gestation. (A) Expression of
CD27/CD28 and perforin by  T cells from a representative CMV-uninfected (bottom) and a representative CMV-infected fetus (Pos4; top), both at the
gestational age of 20–21 wk. Dot plots are presented and numbers indicate the percentages of  T cells negative for CD27 and CD28 and positive for
perforin. CMV, results are representative of 12 (CD27CD28) and 7 (perforin) fetuses (gestation range: 20 wk, 3 d–29 wk, 2 d); CMV+, results are represen-
tative of 13 (CD27CD28) and 5 (perforin) fetuses (gestation range: 20 wk, 5 d–29 wk, 2 d). (B) Spectratyping for CDR31 of four CMV-infected fetuses
for which we had blood samples both at the time of delivery and at earlier gestation times. The enrichment for the CDR31 size of 11 aa consists of the
sequence CALGELGDDKLIF (Table S2).

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in the microarray analysis: CCR5 and CX3CR1 (fractalkine
receptor). Fractalkine can be produced by endothelial cells in
the context of CMV infection (Bolovan-Fritts et al., 2004),
thus possibly attracting differentiated CX3CR1+ fetal 
T cells to the site of infection. Together, our data indicate
that fetal  T cells generated in utero during CMV infec-
tion are equipped with a range of antiviral effector mecha-
nisms, including IFN- production and granule-mediated
cytotoxicity. Indeed,  T cell clones generated from CMV-
infected newborns killed CMV-infected cells and limited
CMV replication in vitro. It is therefore likely that they par-
ticipate in the limitation of the viral spread in the fetus. In
kidney-transplanted patients with acute CMV infection, ex-
pansion of  T cells is associated with the clinical resolu-
tion, suggesting a protective role of the expanded  T cells
(Lafarge et al., 2001; Halary et al., 2005).
We demonstrated that CMV infection during fetal life
leads to the oligoclonal expansion of  T cells, which is
characterized by highly restricted CDR31 and CDR32
repertoires and by the high enrichment of a public CDR31-
CDR38 sequence. Expanded  T cells were negative for
V9 and included V1+, V2+, and V3+ cells. In contrast, in
adult CMV-infected kidney transplanted patients, expanded
 T cells do not include V2+ cells and there is no restric-
tion of CDR32 (Déchanet et al., 1999). V9V2+ 
T cells are very rare in the adult (Morita et al., 1994), pro-
viding a possible explanation of why Déchanet et al. (1999)
did not detect any expansion of this subset in adults (Pitard
et al., 2008).
TCR- chains have the highest potential diversity in the
CDR3 loop (1016 combinations) among all antigen recep-
tor chains (TCR-, TCR-, TCR-, TCR-, IgH, and IgL)
because multiple D gene segments can join together, all D
gene segments can be read in all three open reading frames,
and N nucleotides can be inserted into the junctions of each
of the joining segments (Chien and Konigshofer, 2007).
Therefore, it was surprising to identify a high enrichment of
exactly the same CDR31 sequence (i.e., public CDR3) in
all fetuses with differentiated V1+  T cells upon congeni-
tal CMV infection (ELGDD). It has been suggested that
much of the diversity of the CDR3 junctions of the  chain
may confer different affinities of the  TCR rather than the
ability to recognize different ligands (Chien and Konigshofer,
2007). In addition, in adult CD8  T cells, public CMV-
reactive TCR sequences bind the MHC–peptide complexes
with higher affinity than private MHC peptide–specific TCR
sequences (Trautmann et al., 2005; Day et al., 2007). This
suggests that the public CDR31-ELGDD is enriched by
recognition of a CMV-induced ligand with high affinity.
Our results show for the first time, to our knowledge, the
expansion of a public  TCR CDR3 in the context of an
infection. Furthermore, we demonstrated that the public
CDR31 pairs with a public CDR38 sequence (DTTGW),
indicating that both the  and  chain are important for the
recognition of the putative ligand. In addition, this public
V8V1 TCR showed reactivity against CMV-infected
(Table S4; CMV-infected newborn Pos4). Thus, despite the
recent description of selective impairments of  T cells in
preterm infants (Gibbons et al., 2009),  T cell are able to
develop robust responses toward CMV infection in utero at
as early as 21 wk of gestation.
DISCUSSION
In this study, we demonstrate that CMV infection in utero
leads to the oligoclonal expansion and differentiation of fetal
 T cells, which express high levels of NKR and cytotoxic
mediators and produce IFN-. Both activating (e.g., activating
KIR, NKG2C, and NKG2D) and inhibitory (e.g., inhibitory
KIR, NKG2A, and KLRG1) NKRs were highly expressed
in  T cells derived from congenitally infected newborns.
This would allow them to sense CMV-induced changes in
infected target cells; HLA-E (ligand for NKG2A/NKG2C)
expression is increased upon CMV infection, whereas classi-
cal MHC class I (ligands for KIR) expression is decreased
(Wilkinson et al., 2008). In comparison with conventional
T cells, it has been described that adult  T cells express
high levels of NKR, like members of the C-type lectin and
the KIR family (Battistini et al., 1997; De Libero, 1999;
Pennington et al., 2005). We confirmed the expression of
KLRG1 on  T cells in CMV-uninfected newborns (Eberl
et al., 2005) and also showed that NKG2D is constitutively
expressed. In contrast, unlike NK cells (Dalle et al., 2005),
other NKRs (CD94/NKG2x and KIR family members)
were not expressed or were expressed at very low levels on
 T cells from CMV-uninfected newborns. Thus the ma-
jority of NKR expression on adult  T cells is likely to be
the consequence of infections after birth. CMV infection in
utero induced the up-regulation of various cytotoxic media-
tors in fetal  T cells, including almost all members of the
granzyme family, perforin, granulysin, FasL, and TRAIL.
Perforin and granulysin are membrane-disrupting molecules
and most granzymes have been shown to be involved in
killing of target cells, with most evidence for granzyme B
(Lieberman, 2003; Chowdhury and Lieberman, 2008). In ad-
dition, other granzyme-mediated antiviral mechanisms have
been recently described: granzyme A plays a proinflamma-
tory role (Metkar et al., 2008), granzyme M targets -tubulin
(Bovenschen et al., 2008), and granzyme H cleaves La, a
phosphoprotein involved in cellular and viral RNA metabo-
lism (Romero et al., 2009). It is of note that granzyme
H cleaves an adenovirus-encoded granzyme B inhibitor
(Andrade et al., 2007). Analysis of the profile of cytokine
genes expressed in fetal  T cells derived from CMV-infected
newborns revealed the restricted high expression of IFN-.
In parallel, we detected elevated levels of the T-box tran-
scription factors T-bet and eomes, which are involved in
the rapid and vigorous IFN- production by  T cells (Yin
et al., 2002; Chen et al., 2007). In contrast, expression of
other transcription factor genes like GATA3 (Th2) or ROR-t
(Th17) was not affected, coinciding with the absence of
modulation of cytokine genes associated with these Th sub-
sets. Only two chemokine receptors were significantly increased

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Antiviral  T cells in utero | Vermijlen et al.
newborns and 22 uninfected control newborns as well as 13 infected and 12
uninfected fetuses. Symptomatic congenital infection was diagnosed in fetus
Pos12 who had brain lesions at antenatal magnetic resonance imaging and an
abnormal postnatal neurological development and in fetus Pos5 who had an
abnormal postnatal neurological development.
Flow cytometry. The following antibodies were used: CD3–pacific blue
(clone SP34-2), -PE (11F2), -FITC (11F2), CD27-APC (L128),
CD27-FITC (L128), CD94-APC (HP-3D9), CD158a-FITC (HP-3E4),
CD158b-FITC (CH-L), HLA-DR-APC-Cy7 (L243), NKG2D-APC
(1D11), perforin-FITC (G9), granzyme A–FITC (CB9), Ki-67–FITC
(B56), and IFN-–FITC (25723.11; BD); V2-FITC (IMMU389),
NKG2A-PE (Z199), CD3-ECD (UCHT1), and CD28-ECD (CD28.2;
Beckman Coulter); V1-FITC (TS1; Thermo Fisher Scientific); NKG2C-
APC (134591; R&D Systems); CX3CR1-PE (2A9-1; MBL International);
and T-bet–PE (4B10; eBioscience). V5-PC5 (IMMU360) and V3-FITC
(P11.5B) were derived from Beckman Coulter via custom design service.
V2/3/4-biotin and V2/3/4-FITC (23D12), V4-FITC, V5/3-biotin
(56.3), and V8-biotin (R4.5.1) were provided by D. Wesch (Institute of
Immunology, University of Kiel, Kiel, Germany; Kabelitz et al., 1994; Hinz
et al., 1997; Wesch et al., 1998). KLRG1–Alexa Fluor 488 (13F12F2) was
provided by H. Pircher (University of Freiburg, Freiburg, Germany;
Marcolino et al., 2004) and unlabeled V3 antibody by E. Scotet (Institut
National de la Santé et de la Recherche Médicale U601, Nantes, France;
Peyrat et al., 1995). Staining was done on whole blood. Red blood cells were
lysed using FACS Lysing solution (BD). The absolute number of 
T cells in whole blood was determined using Trucount beads (BD). Intracel-
lular staining for perforin-FITC, granzyme A–FITC, and Ki-67–FITC was
performed with the Perm 2 kit (BD) and for T-bet–PE with the Foxp3
staining buffer set (eBioscience). For the detection of IFN-, PBMCs were
stimulated for 4 h with 10 ng/ml PMA and 2 µM ionomycin in the presence
of 2 µM monensin. Staining was done using the Cytofix/Cytoperm kit (BD).
Cells were run on the CyAn flow cytometer equipped with three lasers (405,
488, and 633 nm) and data were analyzed using Summit 4.3 (Dako).
Microarray analysis. PBMCs were isolated from cord blood by Lympho-
prep gradient centrifugation (Axis-Shield). After depletion of remaining red
blood cells and CD4+ cells by magnetic cell sorting (Miltenyi Biotec),
CD3++ lymphocytes were sorted till high purity (>99%) with a MoFlo
sorter (Dako). The  T cell yield varied from 80,000–300,000 cells per cord
blood sample. Total RNA was isolated using the RNeasy Micro kit
(QIAGEN) from sorted  T cells derived from three CMV-infected
newborns and three CMV-uninfected newborns. RNA concentration was
measured using the NanoDrop (Thermo Fisher Scientific) and RNA quality
was assessed using the Bioanalyzer 2100 (Agilent Technologies). RNA was
amplified into biotin-labeled complementary RNA (cRNA) by one-round
in vitro transcription using the Premier kit (Applied Biosystems). The cRNA
was fragmented and hybridized on the Human Genome U133 Plus 2.0
GeneChip (Affymetrix). Staining and scanning was done on the Affymetrix
platform. The procedures, from RNA quality control to generation of raw
data (CEL files), were performed at DNAVision (Gosselies, Belgium). The
raw data were analyzed using the Affy package of Limma (linear models for
microarray data; www.bioconductor.org), including fitting a linear model
(lmfit) as described previously (Vermijlen et al., 2007). M- and A-values for
each gene were generated. M (log2 of the fold change) is related to the de-
gree of differential expression between the  T cells from CMV-infected
newborns versus  T cells from CMV-uninfected newborns, whereas A is
a measurement of the mean signal intensity. Genes were regarded as differ-
entially expressed if the absolute M-value was >0.5 with a p-value <0.05.
Genes with M-values >0.5 are enriched in the  T cells derived from
CMV-infected newborns, whereas genes with M-values <0.5 are enriched
in the  T cells derived from CMV-uninfected newborns. The Database
for Annotation, Visualization and Integrated Discovery (DAVID; http://
david.abcc.ncifcrf.gov/) was used to assist in the discovery of functionally
related groups of differentially expressed genes. Microarray data and procedures
target cells in vitro. It is of note that both the CDR31-
ELGDD and CDR38-DTTGW were germline encoded, as
the CDR31 was only formed by the V1 gene, one D
gene (D3) and the J1 gene, and the CDR38 by the V8
gene and the JP1 gene, without any addition of P/N nucle-
otides. Similarly, the mouse T22/T10-binding CDR3 does
not contain N nucleotides (Adams et al., 2005; Chien and
Konigshofer, 2007). This contrasts highly with  T cells,
where the most critical amino acids in the CDR3 and
CDR3 involved in the recognition of the MHC–peptide
complex are encoded either completely or partially by N nu-
cleotides (Davis et al., 1998; Chien and Konigshofer, 2007).
Despite the immaturity of the neonatal immune system
and possible mechanisms of immunosuppression by regula-
tory T cells (Mold et al., 2008), CMV infection is efficient in
stimulating vigorous responses of both  T cells and CD8
 T cells (Marchant et al., 2003) during fetal life. Studies in
mice show that the protective role of  T cells in early life
is not dependent on  T cells (Ramsburg et al., 2003).
Conversely, in a model of West Nile virus infection, it has
been shown that  T cells facilitate the CD8  T cell re-
sponse (Wang et al., 2006). In comparison with adult DC,
fetal DC shows impaired functions (Goriely et al., 2004;
Levy, 2007). Because  T cell differentiation is not depen-
dent or is less dependent on DC in comparison with 
T cells, it is reasonable to believe that fetal DC defects would
not prevent  T cell differentiation upon fetal exposure to
CMV. Instead,  T cells may recognize their ligands directly
on infected cells in tissues (Hayday, 2009). Such activated
fetal  T cells could, in turn, induce fetal DC maturation
(Ismaili et al., 2002; Conti et al., 2005; Caccamo et al., 2006;
Devilder et al., 2006; Eberl et al., 2009) and/or directly
activate naive CD8  T cells via antigen cross-presentation
(Brandes et al., 2009), as shown in adult cells. Such mech-
anisms could contribute to the development of functional
CD8  T cell responses to CMV infection during fetal life
(Marchant et al., 2003).
We conclude that human  T cells can mount a vigor-
ous response to CMV infection during development in utero,
providing an important mechanism by which the fetus can
fight pathogens. Identification of the  TCR ligands in-
duced upon CMV infection, like the putative ligand of the
public V8V1 TCR, will likely be useful to design novel
vaccination strategies against viral infection in early life.
MATERIALS AND METHODS
Study population. This study was approved by the Hôpital Erasme and
Hôpital Saint-Pierre ethical committees. Women with suspected primary
CMV infection were referred to the Fetal Medicine Units of the Hôpital
Erasme or Hôpital Saint-Pierre. Diagnosis of primary maternal infection was
based on anti-CMV IgG seroconversion or on the detection of high titers of
anti-CMV–specific IgM, as described previously (Liesnard et al., 2000).
After maternal informed consent, 20–50 ml of cord blood was collected at
birth (full term, >37 wk gestation). In some cases, fetal cord blood was
collected at earlier gestation ages (1 ml by cordocentesis). Diagnosis of
congenital infection was based on the detection of CMV genome by PCR
and/or by viral culture on amniotic fluid and/or on newborn urine collected
during the first week of life. The study included 19 CMV-infected

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819
30,000 cells/well and, after 6 h, supernatant was collected. Release of IFN-
into the supernatant was quantified by ELISA (Invitrogen). The killing assay
was performed as described previously (Vermijlen et al., 2002) with some
modifications. Fibroblasts with [methyl-3H]thymidine-labeled DNA were
infected with CMV for 5 d and coincubated with  T cells at the indicated
effector to target ratios. After 4 h of coincubation, the level of DNA frag-
mentation induced by the  T cells in the fibroblasts was determined as
previously described (Vermijlen et al., 2002).
CMV replication assay. Confluent monolayers of human embryonic lung
fibroblasts (HEL299) in flat-bottom 96-well plates were incubated with
CMV (TB40/E) for 2 h (MOI 0.1), washed, and incubated with medium
alone, with a public V8V1 clone or with a control V9V2 clone (150,000
cells per well). After 7 d, the quantity of infectious CMV from the superna-
tant was determined in quadruplicate by standard plaque assay titration
(in plaque forming units).
Statistical analysis. Differences between CMV-infected newborns and
CMV-uninfected newborns were determined using the nonparametric
Mann-Whitney test using InStat software (GraphPad Software, Inc.). Differ-
ences were regarded as significant at P < 0.05.
Online supplemental material. Fig. S1 shows the V chain expression
of CMV-infected and CMV-uninfected newborns. Fig. S2 shows the as-
sociation of the late differentiation phenotype of  T cells with the expres-
sion of cytotoxic mediators and chemokine receptor CX3CR1. Fig. S3 shows
the CDR32 repertoire. Fig. S4 shows TCR/CD3-dependent IFN-
production by more public V8V1 clones upon coincubation with
CMV-infected target cells. Table S1 provides an overview of differen-
tially expressed genes in  T cells from CMV-infected newborns versus
CMV-uninfected newborns. Tables S2–S4 contain the sequencing data for
CDR31, CDR32, and CDR38 of CMV-infected and CMV-uninfected
newborns. Online supplemental material is available at http://www.jem
.org/cgi/content/full/jem.20090348/DC1.
We are grateful to all the mothers for participating in this study. We would like to
thank Sandra Lecomte for sample and data management, Frederic Lhommé for cell
sorting, Muriel Stubbe and Binita Dutta for fruitful discussions, and Julie Déchanet-
Merville, Bart Vandekerckhove, and Yasmin Haque for tips on cloning T cells. We
are grateful to Daniela Wesch for providing antibodies directed against different
V chains of the VI family, and we would like to thank Hanspeter Pircher for the
KLRG1–Alexa Fluor 488 antibody, Emmanuel Scotet for the unlabeled V3 antibody,
and Zsuzsanna Tabi for providing the CMV strain TB40/E.
This work was supported by the Belgian Science Policy (return grant to
D. Vermijlen and Interuniversity Attraction Pole), European Commission (FP6),
National Fund for Scientific Research (FNRS), Government of the Walloon Region,
and GSK Biologicals. A. Marchant is a senior research associate of the FNRS.
The authors have no conflicting financial interests.
Submitted: 13 February 2009
Accepted: 2 March 2010
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CMV-infected newborns and CMV-uninfected newborns, after which
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PCR (40 cycles) was performed with C (5-GTAGAATTCCTTCAC-
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