CXCR5 identifies a subset of Vgamma9Vdelta2 T cells which secrete IL-4 and IL-10 and help B cells for antibody production.
ABSTRACT Vgamma9Vdelta2 T lymphocytes recognize nonpeptidic Ags and mount effector functions in cellular immune responses against microorganisms and tumors, but little is known about their role in Ab-mediated immune responses. We show here that expression of CXCR5 identifies a unique subset of Vgamma9Vdelta2 T cells which express the costimulatory molecules ICOS and CD40L, secrete IL-2, IL-4, and IL-10 and help B cells for Ab production. These properties portray CXCR5+ Vgamma9Vdelta2 T cells as a distinct memory T cell subset with B cell helper function.
- SourceAvailable from: healthcare.uiowa.eduSpringer Seminars in Immunopathology 01/2000; 22(3):191-217. · 4.17 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Most human peripheral blood gamma delta T lymphocytes respond to hitherto unidentified mycobacterial antigens. Four ligands from Mycobacterium tuberculosis strain H37Rv that stimulated proliferation of a major human gamma delta T cell subset were isolated and partially characterized. One of these ligands, TUBag4, is a 5' triphosphorylated thymidine-containing compound, to which the three other stimulatory molecules are structurally related. These findings support the hypothesis that some gamma delta T cells recognize nonpeptidic ligands.Science 05/1994; 264(5156):267-70. · 31.03 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: T lymphocytes express either alpha beta or gamma delta T-cell receptor heterodimers. Most alpha beta T cells recognize antigenic peptides bound to major histocompatibility complex molecules but the antigen recognition and biological function of gamma delta T cells is unknown. A major human gamma delta T-cell subset expressing V gamma 2 and V delta 2 germline genes, but having diverse junctional sequences, is found in human mycobacterial lesions and responds in vitro to antigens of bacteria and parasites. In addition, certain haematopoietic tumour cells are specifically recognized and lysed by these T cells. V gamma 2V delta 2-bearing T cells were shown to recognize mycobacterial antigens that are protease resistant and phosphatase sensitive. Because of the difficulty in isolating natural antigens from mycobacterial culture filtrates or extracts, we synthesized a series of monoalkyl phosphates, and found that some, particularly monoethyl phosphate, could mimic the activity of mycobacterial antigens in stimulating these gamma delta T cells. Here we report the identification of natural antigens produced by mycobacteria recognized by human V gamma 2V delta 2-bearing T cells as isopentenyl pyrophosphate and related prenyl pyrophosphate derivatives, compounds involved in the synthesis of complex polyisoprenoid compounds in microbial and mammalian cells. Substitution of phosphate for the pyrophosphate moiety, or elimination of the double bond, greatly reduced antigenic activity of these compounds. These results provide formal evidence that, in contrast to recognition of major histocompatibility complex-bound peptide antigens by alpha beta T cells, human gamma delta T cells can recognize naturally occurring small non-peptidic antigens.Nature 06/1995; 375(6527):155-8. · 38.60 Impact Factor
CXCR5 Identifies a Subset of V?9V?2 T Cells which Secrete
IL-4 and IL-10 and Help B Cells for Antibody Production1
Nadia Caccamo,* Luca Battistini,†Marc Bonneville,‡Fabrizio Poccia,§Jean Jacques Fournie ´,¶
Serena Meraviglia,* Giovanna Borsellino,†Richard A. Kroczek,?Carmela La Mendola,*
Emmanuel Scotet,‡Francesco Dieli,2* and Alfredo Salerno*
V?9V?2 T lymphocytes recognize nonpeptidic Ags and mount effector functions in cellular immune responses against microor-
ganisms and tumors, but little is known about their role in Ab-mediated immune responses. We show here that expression of
CXCR5 identifies a unique subset of V?9V?2 T cells which express the costimulatory molecules ICOS and CD40L, secrete IL-2,
IL-4, and IL-10 and help B cells for Ab production. These properties portray CXCR5?V?9V?2 T cells as a distinct memory T
cell subset with B cell helper function. The Journal of Immunology, 2006, 177: 5290–5295.
V?2 chains which directly recognize nonpeptide ligands without
presentation by MHC molecules (1). The nonpeptide ligands, re-
ferred to as phosphoantigens, comprise isoprenoid pathway me-
tabolites derived from mycobacteria (2–5) and tumor cells (6).
Accordingly, pharmacological agents promoting accumulation of
such metabolites, such as aminobisphosphonates (7), sensitize
cells to V?9V?2 T cell recognition. Moreover, it has been recently
found that ATPase expressed on tumor cell surface promotes tu-
mor recognition by V?9V?2 T cells (8).
V?9V?2 T lymphocytes are heterogeneous and comprise dis-
tinct populations that can be distinguished on the basis of surface
markers expression, effector functions, and migratory properties:
naive(CD45RA?CD27?) and centralmemory (TCM
CD45RA?CD27?) cells home to secondary lymphoid organs
and lack immediate effector functions, while effector memory
(CD45RA?CD27?) cells home to sites of inflammation where
they display immediate effector functions such as cytokine pro-
duction and cytotoxicity, respectively (9). Based on their effector
properties, V?9V?2 T lymphocytes are supposed to play an im-
portant role in cellular immune responses against intracellular mi-
minor T cell population, ?? T cells have a unique pattern
of Ag recognition. In humans, the vast majority of cir-
culating ?? T cells express a TCR comprised of V?9 and
croorganisms and tumors (10). However, whether V?9V?2 T lym-
phocytes also participate in Ab-mediated immune responses
remains unclear. Earlier pioneering studies in ?? T cell-deficient
mice demonstrated a nonredundant role for ?? T cells in the gen-
eration of antimicrobial Abs (11, 12) and autoantibodies (13–15),
but as ?? T cell-deficient mice did not show marked defects in IgM
and IgG production, ?? T cells may have a modulatory, rather than
a primary function in the control of humoral immunity. Ab pro-
duction was also increased in in vitro cultures of human ?? T cells
with B cells (16, 17), but the amount of secreted Ab was low and
the mechanisms underlying the observed B cell help were not ex-
amined. A more recent study (18) has shown that human ?? T cells
are found in the follicles of secondary lymphoid organs, express
costimulatory molecules after TCR triggering, and provide B cell
help in vitro, but the intrafollicular ?? T cell subset responsible for
such an activity was not identified.
We show here that expression of CXCR5 defines a subset of
peripheral blood V?9V?2 cells which upon Ag stimulation express
the costimulatory molecules ICOS and CD40L, secrete IL-4 and
IL-10, and provide B cell help for Ab production in vitro.
Materials and Methods
PBMC were obtained from the heparinized blood of 15 healthy volunteers
(9 males, 6 females, age range 20–23 years). PBMC and tonsils were
obtained from seven individuals undergoing tonsillectomy (four males,
three females, age range 9–14 years). All individuals gave informed con-
sent to participate to this study.
FACS staining and sorting
PBMC were isolated from heparinized blood or tonsils by Ficoll-Hypaque
(Pharmacia Biotech). The following conjugated Abs were used in different
combinations: anti-V?2 (Coulter), anti-V?9 (Coulter), anti-CD27 (BD
Pharmingen), anti-CD45RA (Coulter), anti-CD45RO (Coulter), anti-CD3
(Sigma-Aldrich), anti-CD25 (BD Pharmingen), anti-CD62L (BD Pharm-
ingen), anti-CCR7 (a gift of Dr. M. Lipp, Max-Delbruch-Center for Mo-
lecular Medicine, Berlin, Germany), anti-HLA DR monomorphic (a gift of
Prof. V. Horejsi, Institute of Molecular Genetics, Academy of Science of
the Czech Republic, Prague, Czech Republic), anti-CCR5 (BD Pharmin-
gen), anti-CXCR3 (BD Pharmingen), anti-ICOS, anti-CD40L (BD Pharm-
ingen), and anti-CXCR5 (R&D Systems). Data were acquired on a FAC-
SCalibur or a FACSCanto instruments (BD Biosciences) and analyzed
using CellQuest software (BD Immunocytometry Systems) or FlowJo
(Tree Star). Tonsillar B cells were isolated by use of CD19 microbeads
(Miltenyi Biotec), according to the manufacturer’s instruction. For isola-
tion of peripheral blood naive B cells, the procedure described in Ref. 19
*Dipartimento di Biopatologia e Metodologie Biomediche, Universita ` di Palermo,
Palermo, Italy;†Neuroimmunology Unit, Istituto di Ricovero e Cura a Carattere Sci-
entifico Santa Lucia Foundation, Rome, Italy;‡Institut National de la Sante ´ et de la
Recherche Me ´dicale (Inserm) Unite ´ 601, Institut de Biologie, Nantes, France;§Lab-
oratory of Immunology, National Institute for Infectious Diseases “L. Spallanzani,”
Rome, Italy;¶Inserm, Unite ´ 563, Centre de Physiopathologie de Toulouse Purpan,
Toulouse, France; and?Molecular Immunology, Robert Koch Institute, Berlin, Ger-
Received for publication May 9, 2006. Accepted for publication July 28, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by grants from the Commission of the European Union,
6FP, contract LSHP-CT-2003-503367, TB-VAC. F.D. was supported by grants from
the University of Palermo.
2Address correspondence and reprint requests to Dr. Francesco Dieli, Dipartimento
di Biopatologia e Metodologie Biomediche, Universita ` di Palermo, Corso Tukory
211, Palermo 90134 Italy. E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: TCM, central memory T; TEM, effector memory T;
BrHPP, bromohydrinpyrophosphate; ThF, follicular B Th cell.
The Journal of Immunology
Copyright © 2006 by The American Association of Immunologists, Inc.0022-1767/06/$02.00
was used. Briefly, CD19?cells were first isolated by the use of CD19
microbeads and were labeled with 25 nM MitoTracker Green FM (Molec-
ular Probes), a substrate for ABCB1, for 25 min at 37° C and washed twice.
Followingstaining forCD27 and
(CD19?CD27?IgG?ABCB1?) were sorted by FACSVantage (BD Bio-
sciences) with a purity of 99%. Different subsets of V?9V?2 T cells were
sorted similarly by FACSVantage (BD Biosciences).
Cytokine production and chemotaxis assay
The medium used throughout was complete RPMI 1640 medium (Invitro-
gen Life Technologies) supplemented with 10% heat-inactivated FCS (In-
vitrogen Life Technologies), 2 mM L-glutamine, 20 nM HEPES and 100
U/ml penicillin/streptomycin. Sorted V?2 cell subsets were cultured at
37°C, in the presence of 5% CO2, at 105/ml in 96-well flat-bottom plates
(0.2 ml/well), with different concentrations of bromohydrinpyrophosphate
(BrHPP) in the presence of irradiated (5000 rad from a cesium source)
allogeneic dendritic cells (20). IFN-?, TNF-?, IL-2, IL-4, and IL-10 levels
in the 24-h culture supernatants were assessed by two mAbs sandwich
ELISA following manufacturer’s recommendations (R&D Systems).
The chemotactic ability of CXCR5?V?9V?2 cells was assayed using a
double-chamber system with 3-?m pores (Transwell; Costar), according to
Ref. 21. Briefly, 105sorted CXCR5?or CXCR5?V?9V?2 cells were
added to the upper chamber and CXCL13 (recombinant human CXCL13,
BCA-1; R&D Systems, 3 ?M final concentration) to the lower chamber
and incubated at 37°C for 2 h in a 5% CO2humidified incubator. In some
experiments, anti-CXCR5 or isotype control mAbs were added to the lower
chamber during the test. Assays were performed in triplicate. Afterward,
the membrane was removed, washed on the upper side with PBS, fixed, and
stained. Migrated cells were counted microscopically at ?1000 magnifi-
cation in five randomly selected fields per well. Percentage migration was
calculated by measuring the counts recovered from the lower chamber and
comparing them to the total input counts; results represent the mean ? SD
of three independent experiments.
Ab production in vitro
V?9V?2 T cell help in Ab production was studied as follows. Different
subsets of peripheral blood V?9V?2 T cells were sorted by FACS and
cocultured with sorted tonsillar B cells or peripheral blood-derived naive B
cells, in 96-well plates at 105cells/well each of T and B cells in the pres-
ence or absence of BrHPP for 10 days. IgM, IgG, and IgA levels in the
culture supernatants were determined by ELISA. When naive B cells were
used, F(ab?)2of goat anti-human Ig (Jackson ImmunoResearch Laborato-
ries) was added to cultures as a BCR trigger, at the final concentration of
2 ?g/ml. Anti-CD40L mAb and ICOS ligand fused to the Fc portion of
mouse IgG2a (ICOSL-muIg; both reagents from Alexis through Vinci-
Biochem, Firenze, Italy), neutralizing anti-human IL-4 and IL-10 (BD Bio-
sciences) and isotype matched control mAbs, were added at the beginning
of cultures at the final concentration of 15 ?g/ml. As a control of the
ICOSL-muIg, we used mouse IL-7 fused to the Fc portion of mouse IgG2a
(a gift from Prof. G. Stassi, Department of Surgical and Oncological Sci-
ence, University of Palermo, Palermo, Italy).
The double-tailed Student t test was used to analyze the significance of
differences between groups.
Expression of CXCR5 on human V?9V?2 T cells
Expression of the chemokine receptor CXCR5 defines a popula-
tion of CD4 Th cells which support the production of Igs (21, 22).
CXCR5 expression has been also detected on human V?9V?2 T
cells by some authors (23), but not by others (18).
CXCR5 expression was studied on peripheral blood V?9V?2 T
cells. In a population of healthy donors (n ? 15, age range 20–23
years), ?15% of total V?2 T cells are CXCR5?(14.6 ? 6.2, range
5.3–46.8, see Fig. 1). In a cohort of seven young patients (age
range 9–14 years) undergoing tonsillectomy the percentage of pe-
ripheral blood CXCR5?V?2 T cells was not significantly different
from in adults (15.8 ? 7.3, range 4.8–39.5, data not shown). How-
ever, CXCR5?V?9V?2 T cells were highly enriched in inflamed
tonsils where they account for about half the size of the V?9V?2
T cell population (Fig. 1).
We have previously reported (9) that the expression of CD45RA
and CD27 Ags defines four subsets of human V?9V?2 T cells with
distinctive functional properties and compartmentalization routes.
FACS analysis demonstrates that the vast majority of peripheral
blood CXCR5?V?9V?2 cells do not express CD45RA, but ex-
press CD27, CD45RO, CCR7, and CD62L (Table I), suggesting
that most of them have a TCMphenotype. Peripheral blood
CXCR5?V?9V?2 T cells do not express the activation markers
CD25 and HLA-DR and also lack expression of the costimulatory
molecules CD40L and ICOS.
In tonsils, CXCR5?V?9V?2 T cells had a TCMphenotype like
their peripheral blood counterpart (see Table I), but most of them
express several activation markers and costimulatory molecules
(CD25, HLA-DR, CD40L and ICOS, see Table I), suggesting that
they are engaged in immune responses occurring in tonsils. More-
over, expression of CCR7, which causes homing to the T cell areas
of secondary lymphoid tissues (24) is found on the vast majority of
peripheral blood CXCR5?V?9V?2 T cells (Table I) but is mark-
edly reduced on tonsillar CXCR5?V?9V?2 T cells (Table I), sug-
gesting the possibility of a ligand-induced CCR7 down-modula-
tion. Similarly, CD62L expression is reduced on tonsillar
CXCR5?V?9V?2 T cells, implying that the majority of tonsillar
CXCR5?V?9V?2 T cells have recently immigrated from circula-
tion. The chemokine receptors CXCR3 and CCR5 were weakly
expressed on tonsillar CXCR5?V?9V?2 T cells, but were not de-
tected on peripheral blood CXCR5?V?9V?2 T cells (Table I).
To assess whether costimulatory molecules expression of tonsil
CXCR5?V?9V?2 T cells was due to Ag-dependent activation, we
sorted CXCR5?and CXCR5?V?9V?2 TCMand V?9V?2 TEM
cells from peripheral blood and cultured in vitro with BrHPP and
irradiated dendritic cells. None of the three subsets stained positive
peripheral blood and tonsils. a, Analysis of the relative proportion of
CXCR5?V?9V?2 T cells in peripheral blood and tonsils. b, FACS analysis
of peripheral blood and tonsils mononuclear cells stained with different
Distribution and phenotype of CXCR5?V?9V?2 T cells in
Table I. Surface markers expression on CXCR5?V?9V?2?T cells in
peripheral blood and inflamed tonsilsa
(n ? 15) Tonsils (n ? 6)
1 ? 2 (4)
98 ? 7 (85)
72 ? 5 (67)
1 ? 0.4 (2)
0.5 ? 0.1 (4)
2.4 ? 0.4 (4)
1.5 ? 0.2 (3)
95 ? 4 (73)
97 ? 3.9 (86)
1 ? 0.1 (5)
2.1 ? 0.6 (7)
2 ? 3 (5)
95 ? 9 (38)
79 ? 7 (51)
65 ? 7 (44)
58 ? 6 (34)
78 ? 9 (84)
98 ? 6 (115)
33 ? 2 (30)
28 ? 5 (25)
15 ? 2 (18)
9.5 ? 3.1 (11)
aPeripheral blood and tonsil mononuclear cells were stained with mAbs to V?2,
CXCR5, and other cell surface markers, and were analysed by FACS. Values indicate
the percentage ? SE of CXCR5?V?2?cells expressing the indicated cell surface
markers. Values in parentheses indicate the mean fluorescence intensity mean value.
5291The Journal of Immunology
for ICOS and CD40L either in the absence or 24 h after Ag stim-
ulation in vitro (data not shown). However, after 48 and 72 h of Ag
stimulation, CXCR5?and, although at a lower extent CXCR5?
V?9V?2 TCMand V?9V?2 TEMcells expressed CD40L, while
ICOS expression was detected only on CXCR5?V?9V?2 TCM
cells (Fig. 2).
Migratory properties and cytokine production of
CXCR5?V?9V?2 T cells
Freshly isolated tonsillar CXCR5?, but not CXCR5?, V?9V?2 T
cells migrated readily in response to CXCL13/BCA-1 (Fig. 3a).
Responsiveness to BCA-1 was enhanced when the cells were cul-
tured in vitro with BrHPP, but by day 3 of culture migration in
response to BCA-1 consistently decreased; this effect was paral-
leled by CXCR5 receptor expression, which uniformly decreased
upon in vitro culture with BrHPP (Fig. 3a). Of note, migration of
tonsillar CXCR5?V?9V?2 T cells in response to CXCL13/BCA-1
was consistently inhibited by anti-CXCR5 mAb (Fig. 3b).
We studied the pattern of cytokine production in the CXCR5?
V?9V?2 T cell subsets after a 24 h stimulation period with BrHPP
CXCR5?V?9V?2 T cell were separated into CD27?and CD27?
cells and their pattern of cytokine production was compared with
that of CXCR5?CD27?(TEM) and CXCR5?CD27?(TCM)
V?9V?2 T cells. As shown in Table II, CXCR5?CD27?V?9V?2
TEMcells produced IFN-? and TNF-?, but neither IL-4 nor IL-10,
thus confirming our previous results (9). CXCR5?CD27?V?9V?2
TCMcells also produced IFN-? and TNF-?, but the amounts of
cytokines were significantly lower than those produced by
V?9V?2 TEMcells. Ag-stimulated CXCR5?CD27?V?9V?2 T
cells had a different cytokine profile: while producing IFN-? and
TNF-? at a similar extent to CXCR5?CD27?V?9V?2 TCMcells,
they produced significantly higher levels of IL-2, IL-4, and IL-10,
when compared with other subsets of V?9V?2 cells. This cytokine
profile was unique to the CXCR5?CD27?subset, as Ag-stimu-
lated CXCR5?CD27?V?9V?2 T cells produced IFN-? and
TNF-?, but not IL-2, IL-4, and IL-10.
CXCR5?V?9V?2 T cells help B cells for Ab production
As CXCR5?V?9V?2 T cells express costimulatory molecules,
produce IL-4 and IL-10, we tested whether or not these cells were
able to support B cells to secrete Igs. Peripheral blood CXCR5?
and CXCR5?V?9V?2 T cells were further sorted into CD27?and
CD27?populations and the four T cell subsets were cultured with
CD19?B cells isolated from the tonsil of the same donor, in the
presence or absence of BrHPP. Fig. 4 shows one typical experiment
of five. B cells produced comparable very low amounts of IgA, IgG
and IgM when cultured for 10 days without V?9V?2 T cells or with
the CXCR5?fractions (i.e., CXCR5?CD27?V?9V?2 TCMand
CXCR5?CD27?V?9V?2 TEMcells). Similarly, very low IgA, IgG,
and IgM levels were detected in cocultures of B cells and
CXCR5?CD27?V?9V?2 T cells. In contrast, coculture of B cells
with CXCR5?CD27?V?9V?2 T cells and BrHPP resulted in an
18-fold increase in the production of IgG, 8-fold increase in the pro-
duction of IgA and 7-fold increase in the production of IgM.
Of note, CXCR5?V?9V?2 T cells from peripheral blood did not
cause significant increase of Ab production in cocultures with B
cells conducted in the absence of BrHPP, but total V?9V?2 T cells
or CXCR5?V?9V?2 T cells from the tonsils were able to induce
significant production of Igs even in the absence of Ag, indicating
that they are well equipped for providing B cell help (Fig. 5). The
B cell helper activity of CXCR5?V?9V?2 TCMcells was strictly
dependent on their provision of both costimulatory molecules and
cytokines, as blocking of CD40L or ICOS caused a drastic reduc-
tion of both Ab and similar inhibition was obtained by addition of
Abs to IL-10 and IL-4 (Fig. 6), even if the latter seems to be
dispensable for IgA production.
Because the source of B cells used in these assays is the in-
flamed tonsil it would seem likely that CXCR5?V?9V?2 T cells
act by amplifying or enhancing Ab secretion by preformed plasma
cules in CXCR5?and CXCR5?V?9V?2 T cell subsets. FACS analysis of
CD40L and ICOS expression on different subsets of peripheral blood
V?9V?2 T cells before (black line) or 48 h (blue line) and 72 h (green line)
after stimulation with BrHPP. Control mAb stainings are shown in red
Activation-dependent expression of costimulatory mole-
CXCR5?(?) or CXCR5?(f) V?9V?2 T cells were cultured with BrHPP for up to 8 days and examined for in vitro migration to CXCL13/BCA-1 (3
?M, final concentration) and CXCR5 expression (E). In b, migration of tonsillar CXCR5?V?9V?2 T cells to CXCL13/BCA-1 was conducted in the
presence of anti-CXCR5 or isotype-matched control mAbs (15 ?g/ml, final concentration). Data are representative of three independent experiments. ?, p ?
0.05 when compared with groups consisting of CXCR5?V?9V?2 T migrating to CXCL13 in the absence of any Ab or in the presence of isotype-matched
Migration to CXCL13/BCA-1 and CXCR5 expression during culture of tonsillar CXCR5?V?9V?2 T cells. In a, freshly isolated tonsillar
5292CXCR5?V?9V?2 T CELLS HELP B CELLS
cells. We therefore investigated whether CXCR5?V?9V?2 T cells
can drive naive B cells to become Ab-secreting plasma cells. To
this end, peripheral blood CXCR5?V?9V?2 T cells were cultured
with highly purified naive B cells isolated from the peripheral
blood of the same donors, in the presence or absence of BrHPP and
F(ab?)2of goat anti-human Ig as a BCR trigger, and Ab production
was assessed 10 days later.
As shown in Fig. 7 (one typical experiment of three),
CXCR5?V?9V?2 T cells provided help to naive B cells to pro-
duce IgM, IgG, and IgA Abs, thus indicating that this T cell subset
is also able to provide help.
Our study provides evidence of a direct role for ?? T cells in the
control of humoral immune response and allows us to assign this
property to a subsets of V?9V?2 T cells that express CXCR5.
Circulating CXCR5?V?9V?2 T cells uniformly coexpress
CD45R0, CCR7, and CD62L, but do not express either activation
(CD25 and HLA-DR) or costimulatory (CD40L and ICOS) mol-
ecules. This subset was heterogeneous in terms of CD27 expres-
sion, with the majority (70%) being CD27?. Thus, peripheral
CXCR5?V?9V?2 T cells likely represent a subpopulation of TCM
cells and, thus, differ from effector memory V?9V?2 T cells,
which are proposed to directly participate in immune responses at
inflammatorysites (9). Accordingly,
V?9V?2 T cells lack chemokine receptors, such as CCR5 and
CXCR3, that are typically involved in recruitment of effector
memory T cells to inflammatory sites in peripheral tissues. This
chemokine receptor profile suggests that CXCR5?V?9V?2 T cells
home preferentially into secondary lymphoid tissues and, there-
fore, CXCR5?V?9V?2 T cells from tonsils were selected for fur-
Practically 50% V?9V?2 T cells in tonsils are CXCR5?and the
majority express activation markers (CD69 and HLA-DR) and co-
stimulatory molecules (CD40L and ICOS) suggesting their en-
gagement in B cell activation. Drastic reduction in cell surface
CCR7 and CD62L expression indicates that the majority of local
CXCR5?V?9V?2 T cells have recently entered the tonsils.
Tonsillar CXCR5?V?9V?2 T cells readily migrate in response
to CXCL13/BCA-1, and the response was consistently inhibited by
anti-CXCR5 mAb demonstrating that CXCR5 is functional.
However, both CXCR5 expression and responsiveness to
CXCL13/BCA-1 are sensitive to V?9V?2 T cell activation: in fact,
they increased upon phosphoantigen activation, but by day 3 they
consistently decrease. The requirements for induction of CXCR5
expression in V?9V?2 T cells are not defined. Preliminary data
indicate that phosphoantigen stimulation in the presence or ab-
sence of IL-2 are not sufficient. In mice, CXCR5 expression during
primary responses was shown to depend on sequential signaling by
CD28 and OX40, suggesting the requirement for APCs (25, 26).
Expression of CXCR5 on human V?9V?2 T cells is a matter of
debate. While Kabelitz and coworkers (23) found this receptor
being expressed by a subset of V?9V?2 T cells, Moser and col-
leagues (18) did not detect CXCR5 expression on both peripheral
blood and tonsillar V?9V?2 T cells. Moreover, Forster et al. (27)
found that in healthy individuals, 2% of peripheral blood ?? T
cells, but ?23% of tonsillar ?? T cells express BLR1 (presumably
CXCR5) and this percentage consistently increased in HIV-in-
fected individuals. We have no obvious explanation for the
duction. Tonsillar B cells were cultured alone or in the presence of equal
numbers of the CXCR5?CD27?, CXCR5?CD27?, CXCR5?CD27?, and
CXCR5?CD27?subsets of V?9V?2 T lymphocytes, in the presence of
BrHPP. Ten days later, total IgG, IgA, and IgM levels in culture supernatants
were assessed by ELISA. One of five different experiments is shown. ?, p ?
0.001 when compared with the group consisting of B cell cultured with
CXCR5?V?9V?2 T cells help tonsillar B cells for Ab pro-
lar B cells were cultured alone (Nil) or with peripheral blood- or tonsillar-
derived total V?9V?2 T lymphocytes or their CXCR5?subset, in the pres-
ence or absence of BrHPP. Ten days later, total IgG and IgA levels in
culture supernatants were assessed by ELISA. One of three different ex-
periments is shown. ?, p ? 0.001 and ??, p ? 0.01 when compared with
the group consisting of B cell cultured with medium (Nil).
Ag requirement for CXCR5?V?9V?2 T cell help. Tonsil-
Table II. Cytokine production by CXCR5?and CXCR5?V?9V?2 T cellsa
V?9V?2 T Cell Subset
100 ? 20
150 ? 40
550 ? 65c
600 ? 25c
240 ? 30c
110 ? 20
160 ? 15
10 ? 5
10 ? 10
10 ? 5
250 ? 60
200 ? 55
40 ? 10
10 ? 5
10 ? 5
1400 ? 320b
2300 ? 450b
20 ? 10
50 ? 5
10 ? 5
aPeripheral blood CXCR5?and CXCR5?V?9V?2 T cells were separated into CD27?and CD27?cells and equal numbers
of each of the four subset were cultured with BrHPP. Cytokine levels were assessed by ELISA and data expressed as picrograms
per milliliter ? SD. Similar results were obtained in three independent experiments.
bA value of p ? 0.005 andcp ? 0.001 when compared to values in all other groups.
5293The Journal of Immunology
discrepancy in CXCR5 expression on peripheral blood T cells
between these studies but the results report here clearly demon-
strate CXCR5 expression by a subset of human V?9V?2 T cells.
CXCR5 defines a subset of CD4 memory Th cells which are
now termed follicular B Th (ThF) cells (28). Peripheral blood ThF
cells belong to the subset of TCMcells, i.e., they are resting,
CCR7?and nonpolarized (do not produce Th1/Th2-type cytokines
upon stimulation). Tonsillar ThFcells are also nonpolarized but
differ from peripheral blood ThFcells in that they express a range
of activation markers and costimulatory molecules.
ThFcells are highly efficient at providing B cell help and, thus,
are expected to be well-equipped with costimulatory molecules
and cytokines. In this regard, a significant fraction of tonsillar ThF
cells express CD40L and the majority also express ICOS. How-
ever, the cytokine secretion capacity of tonsillar ThFcells is very
limited and only a subpopulation of germinal center-localized ThF
cells has indeed been identified which produces IL-10.
Differently than CXCR5?CD4????memory T cells, that are
poor producers of cytokines (21, 22), CXCR5?V?9V?2 T cells
have a Th2-type pattern of cytokine production upon Ag stimula-
tion in vitro, as they secrete IL-2, IL-4, and IL-10. This contrasts,
with the cytokine production pattern of the CXCR5?TEMsubsets
of V?9V?2 T cells, which preferentially secrete IFN-? and TNF-?.
Moreover, secretion of IL-4 and IL-10 seems confined to the
CD27?CXCR5?subset of V?9V?2 T cells. The finding of a pop-
ulation of V?9V?2 T cells that secretes IL-4 and IL-10 is not new,
and expands previous results demonstrating IL-4 production by
resting (29–31) and V?9V?2 T cell clones (32, 33), most of which
express CD27 (M. Bonneville and E. Scotet, unpublished
Production of Th2-type cytokines together with expression of
CD40L and ICOSstrongly
CXCR5?V?9V?2 T cells are engaged in B cell activation and help
for Ab production. Interestingly, and differently than CD4?fol-
licular T cells, CD40L, and ICOS are induced late during activa-
tion and persist longer, a finding that may be important for their
function within germinal centers. In fact, ICOS induces production
of various cytokines from recently activated T cells and critically
participates in T cell-dependent immune responses (34) and CD40-
CD40L interaction is an essential step during a T cell-dependent B
cell response as it regulates proliferation of B cells, Ig class switch-
ing, and Ab production (35). Consequently, when coculturing
sorted tonsillar B cells with various subsets of V?9V?2 T cells, we
observed Ig production only within the CXCR5?CD27?fraction,
thus identifying this cell population as the classical helper cells.
Moreover, Ig production was consistently inhibited by blocking
CD40-CD40L and ICOS-ICOSL interactions, or by neutralization
of IL-4 or IL-10. Due to the preactivation status of tonsillar B cells
and that exogenous stimuli were not required for B cell help by
tonsillar V?9V?2 T cells, one may argue that CXCR5?V?9V?2 T
cells may only be active on already activated B cells and hence
during secondary Ab responses. However, the finding that circu-
lating CXCR5?V?9V?2 T cells are also able to help circulating
naive B cells for Ab production, strongly suggests that they play an
important regulatory role in all aspects of humoral immunity.
Contribution of CXCR5?V?9V?2 T cells to Ab-mediated im-
mune responses may occur early during microbial infections, be-
fore full development of acquired responses mediated by CD4 T
cells, which depends on a series of time-consuming steps, includ-
ing Ag uptake and processing by tissue DCs, their relocation to
draining lymph nodes, and T cell priming and effector cell devel-
opment. Accordingly, V?9V?2 T cells induce maturation of my-
eloid dendritic cells (33, 36–38) and, even if for a short period of
time upon Ag activation, may function as APCs themselves (39,
Thus, V?9V?2 T cells may influence the subsequent adaptive
immune responses through interaction with peripheral dendritic
cells and B cells in reactive secondary lymphoid tissues.
We thank Dr. M. Lipp, Prof. V. Horejsi, and Prof. G. Stassi for providing
us with reagents.
The authors have no financial conflict of interest.
1. Morita, C. T., R. Mariuzza, and M. B. Brenner. 2000. Antigen recognition by
human ?? T cells: pattern recognition by the adaptive immune system. Springer
Semin. Immunopathol. 22: 191–217.
2. Constant, P., F. Davodeau, M. A. Peyrat, Y. Poquet, G. Puzo, M. Bonneville, and
J. J. Fournie ´. 1994. Stimulation of human ?? T cells by nonpeptidic mycobac-
terial ligands. Science 264: 267–270.
3. Tanaka, Y., S. Sano, E. Nieves, G. De Libero, D. Rosa, R. L. Modlin,
M. B. Brenner, B. R. Bloom, and C. T. Morita. 1994. Nonpeptide ligands for
human ?? T cells Proc. Natl. Acad. Sci. USA 91: 8175–8179.
4. Tanaka, Y., C. T. Morita, E. Nieves, M. B. Brenner, and B. R. Bloom. 1995.
Natural and synthetic non-peptide antigens recognized by human ?? T cells.
Nature 375: 155–158.
5. Belmant, C., E. Espinosa, F. Halary, I. Apostolou, E. Sicard, M. A. Payrat,
A. Vercellone, P. Kourilsky, G. Gachelin, R. Poupot, et al. 1999. 3-Formyl-1-
butyl pyrophosphate: a novel mycobacterial metabolite-activating human ?? T
cells. J. Biol. Chem. 274: 32079–32084.
Naive B cells were sorted from the peripheral blood as indicated in Ma-
terials and Methods, and were cultured alone or with peripheral blood
CXCR5?V?9V?2 T lymphocytes, in the presence of BrHPP and F(ab?)2
of goat anti-human Ig as a BCR trigger (2 ?g/ml final concentration). Ten
days later, total IgG, IgA, and IgM levels in culture supernatants were
assessed by ELISA. One of three different experiments is shown. ?, p ?
0.001 when compared with all other groups.
CXCR5?V?9V?2 T help naive B cells for Ab production.
stimulatory molecules and cytokines. Cocultures of tonsillar B cells and
peripheral blood CXCR5?V?9V?2 T lymphocytes were conducted as de-
scribed in the legend to Fig. 4, but in the presence of mAbs to costimula-
tory molecules or cytokines (see Materials and Methods). ?, p ? 0.005 and
??, p ? 0.001 when compared with the group consisting of B cells cultured
with CXCR5?V?9V?2 T lymphocytes and BrHPP.
CXCR5?V?9V?2 T-B cell cooperation requires both co-
5294 CXCR5?V?9V?2 T CELLS HELP B CELLS
6. Gober, H. J., M. Kistowska, L. Angman, P. Jeno, L. Mori, and G. De Libero.
2003. Human T cell receptor ?? cells recognize endogenous mevalonate metab-
olites in tumor cells. J. Exp. Med. 197: 163–168.
7. Kunzmann, V., E. Bauer, and M. Wilhelm. 1999. ?? T-cell stimulation by pam-
idronate. N. Eng. J. Med. 340: 737–738.
8. Scotet, E., L. O. Martinez, E. Grant, R. Barbaras, P. Jeno, M. Guiraud,
B. Monsarrat, X. Saulquin, S. Maillet, J. P. Esteve, et al. 2005. Tumor recognition
following V?9V?2 T cell receptor interactions with a surface F1-ATPase-related
structure and apolipoprotein A-I. Immunity 22: 71–80.
9. Dieli, F., F. Poccia, M. Lipp, G. Sireci, N. Caccamo, C. Di Sano, and A. Salerno.
2003. Differentiation of effector/memory V?2 T cells and migratory routes in
lymph nodes or inflammatory sites. J. Exp. Med. 198: 391–397.
10. Bonneville, M., and J. J. Fournie ´. 2005. Sensing cell stress and transformation
through V?9V?2 T cell-mediated recognition of the isoprenoid pathway metab-
olites. Microbes Infect. 7: 503–509.
11. Pao, W., L. Wen, A. L. Smith, A. Gulbranson-Judge, B. Zheng, G. Kelsoe,
I. C. MacLennan, M. J. Owen, and A. C. Hayday. 1996. ?? T cell help of B cells
is induced by repeated parasitic infection, in the absence of other T cells. Curr.
Biol. 6: 1317–1325.
12. Maloy, K. J., B. Odermatt, H. Hengartner, and R. M. Zinkernagel. 1998. Inter-
feron-?-producing ?? T cell-dependent antibody isotype switching in the absence
of germinal center formation during virus infection. Proc. Natl. Acad. Sci. USA
13. Wen, L., S. J. Roberts, J. L. Viney, F. S. Wong, C. Mallick, R. C. Findly, Q. Peng,
J. E. Craft, M. J. Owen, and A. C. Hayday. 1994. Immunoglobulin synthesis and
generalized autoimmunity in mice congenitally deficient in ???T cells. Nature
14. Peng, S. L., M. P. Madaio, D. P. Hughes, N. I. Crispe, M. J. Owen, L. Wen,
A. C. Hayday, and J. Craft. 1996. Murine lupus in the absence of ?? T cells.
J. Immunol. 156: 4041–4049.
15. Wen, L., W. Pao, F. S. Wong, Q. Peng, J. Craft, B. Zheng, G. Kelsoe, L. Dianda,
M. J. Owen, and A. C. Hayday. 1996. Germinal center formation, immunoglob-
ulin class switching, and autoantibody production driven by “non ??” T cells.
J. Exp. Med. 183: 2271–2282.
16. Rajagopalan, S., T. Zordan, G. C. Tsokos, R. M. Lebovitz, and M. W. Lieberman.
1990. Pathogenic anti-DNA autoantibody-inducing T helper cell lines from pa-
tients with active lupus nephritis: isolation of CD4?8?T helper cell lines that
express the ?? T-cell antigen receptor. Proc. Natl. Acad. Sci. USA 87:
17. Horner, A. A., H. Jabara, N. Ramesh, and R. S. Geha. 1995. ?? T lymphocytes
express CD40 ligand and induce isotype switching in B lymphocytes. J. Exp.
Med. 181: 1239–1244.
18. Brandes, M., K. Willimann, A. B. Lang, K. H. Nam, C. Jin, M. B. Brenner,
C. T. Morita, and B. Moser. 2003. Flexible migration program regulates ?? T-cell
involvement in humoral immunity. Blood 102: 3693–3701.
19. Ruprecht, C. R., and A. Lanzavecchia. 2006. Toll-like receptor stimulation as a
third signal required for activation of human naive B cells. Eur. J. Immunol. 36:
20. Caccamo, N., S. Meraviglia, V. Ferlazzo, D. Angelini, G. Borsellino, F. Poccia,
L. Battistini, F. Dieli, and A. Salerno. 2005. Differential requirements for antigen
or homeostatic cytokines for proliferation and differentiation of human V?9V?2
naive, memory and effector T cell subsets. Eur. J. Immunol. 35: 1764–1772.
21. Schaerli, P., K. Willimann, A. B. Lang, M. Lipp, P. Loetscher, and B. Moser.
2000. CXC chemokine receptor 5 expression defines follicular homing T cells
with B cell helper function. J. Exp. Med. 192: 1553–1562.
22. Breitfeld, D., L. Ohl, E. Kremmer, J. Ellwart, F. Sallusto, M. Lipp, and R. Forster.
2000. Follicular B helper T cells express CXC chemokine receptor 5, localize to
B cell follicles, and support immunoglobulin production. J. Exp. Med. 192:
23. Glatzel, A., D. Wesch, F. Schiemann, E. Brandt, O. Janssen, and D. Kabelitz.
2002. Patterns of chemokine receptor expression on peripheral blood ?? T lym-
phocytes: strong expression of CCR5 is a selective feature of V?2/V?9 ?? T
cells. J. Immunol. 168: 4920–4929.
24. Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia. 1999. Two
subsets of memory T lymphocytes with distinct homing potentials and effector
functions. Nature 401: 708–712.
25. Flynn, S., K. M. Toellner, C. Raykundalia, M. Goodall, and P. Lane. 1998. CD4
T cell cytokine differentiation: the B cell activation molecule, OX40 ligand, in-
structs CD4 T cells to express interleukin 4 and upregulates expression of the
chemokine receptor, Blr-1. J. Exp. Med. 188: 297–304.
26. Walker, L. S. K., A. Gulbranson-Judge, S. Flynn, T. Brocker, C. Raykundalia,
M. Goodall, R. Fo ¨rster, M. Lipp, and P. Lane. 1999. Compromised OX40 func-
tion in CD28-deficient mice is linked with failure to develop CXC chemokine
receptor 5-positive CD4 cells and germinal centers. J. Exp. Med. 190:
27. Forster, R., G. Schweigard, S. Johann, T. Emrich, E. Kremmer, C. Nerl, and
M. Lipp. 1997. Abnormal expression of the B-cell homing chemokine receptor
BLR1 during the progression of acquired immunodeficiency syndrome. Blood 90:
28. Vinuesa, C. G., S. G. Tangye, B. Moser, and C. R. Mackay. 2005. Follicular B
helper T cells in antibody responses and autoimmunity. Nat. Rev. Immunol. 5:
29. Wesch, D., A. Glatzel, and D. Kabelitz. 2001. Differentiation of resting human
peripheral blood ?? T cells toward Th1- or Th2-phenotype. Cell Immunol. 12:
30. Ordway, D. J., L. Pinto, L. Costa, M. Martins, C. Leandro, M. Viveiros,
L. Amaral, M. J. Arroz, F. A. Ventura, and H. M. Dockrell. 2005. ?? T cell
responses associated with the development of tuberculosis in health care workers.
FEMS Immunol. Med. Microbiol. 43: 339–350.
31. Ordway, D. J., L. Costa, M. Martins, H. Silveira, L. Amaral, M. J. Arroz,
F. A. Ventura, and H. M. Dockrell. 2004. Increased interleukin-4 production by
CD8 and ?? T cells in health-care workers is associated with the subsequent
development of active tuberculosis. J. Infect. Dis. 190: 756–766.
32. Sireci, G., E. Champagne, J. J. Fournie, F. Dieli, and A. Salerno. 1997. Patterns
of phosphoantigen stimulation of human V?9/V?2 T cell clones include Th0
cytokines. Hum. Immunol. 58: 70–82.
33. Devilder, M. C., S. Maillet, I. Bouyge-Moreau, E. Donnadieu, M. Bonneville, and
E. Scotet. 2006. Potentiation of antigen-stimulated V?9V?2 T cell cytokine pro-
duction by immature dendritic cells (DC) and reciprocal effect on DC maturation.
J. Immunol. 176: 1386–1393.
34. Hutloff, A., A. M. Dittrich, K. C. Beier, B. Eljaschewitsch, R. Kraft,
I. Anagnostopoulos, and R. A. Kroczek. 1999. ICOS is an inducible T-cell co-
stimulator structurally and functionally related to CD28. Nature 397: 263–266.
35. Grewal, I. S., and R. A. Flavell. 1998. CD40 and CD154 in cell-mediated im-
munity. Annu. Rev. Immunol. 16: 111–135.
36. Conti, L., R. Casetti, M. Cardone, B. Varano, A. Martino, F. Belardelli, F. Poccia,
and S. Gessani. 2005. Reciprocal activating interaction between dendritic cells
and pamidronate-stimulated ?? T cells: role of CD86 and inflammatory cyto-
kines. J. Immunol. 174: 252–260.
37. Ismaili, J., V. Olislagers, R. Poupot, J. J. Fournie, and M. Goldman. 2002. Human
?? T cells induce dendritic cell maturation. Clin. Immunol. 103: 296–302.
38. Leslie, D. S., M. S. Vincent, F. M. Spada, H. Das, M. Sugita, C. T. Morita, and
M. B. Brenner. 2002. CD1-mediated ?/? T cell maturation of dendritic cells.
J. Exp. Med. 196: 1575–1584.
39. Moser, B., and M. Brandes. 2006. ?? T cells: an alternative type of professional
APC. Trends Immunol. 27: 112–118.
40. Brandes, M., K. Willimann, and B. Moser. 2005. Professional antigen-presenta-
tion function by human ?? T cells. Science 309: 264–268.
5295The Journal of Immunology