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Blood First Edition
Paper, prepublished
online
June
3, 2004;
DOI 10.1182/blood-2004-01-0331
FcJRIII discriminates between two subsets of VJ9VG2 effector cells
with different responses and activation pathways
Running title: helper and cytolytic effector memory in human γδ T cells
Daniela F. Angelini1, Giovanna Borsellino1, Mary Poupot, Adamo Diamantini, Rémy Poupot,
Giorgio Bernardi, Fabrizio Poccia, Jean-Jacques Fournié, and Luca Battistini
From Neuroimmunology Unit, Santa Lucia Foundation, scientific institute (I.R.C.C.S.), Rome,
Italy (D.F.A., G.B., A.D., L.B.); Départment of Oncogénèse & Signalisation dans les Cellules
Hématopoiétiques, Unité 563 de l’Institut National de la Santé et de la Recherche Médicale,
Centre de Physiopathologie de Toulouse Purpan, Toulouse, France (M.P., R.P., J-J.F.);
Department of Neuroscience, University of Rome “Tor Vergata”, Rome, Italy (G.B.); and
Laboratory of Immunopathology, Padiglione Del Vecchio, National Institute for Infectious
Diseases, Rome, Italy (F.P.)
1
These authors contributed equally to the work.
Supported by institutional grants from the Italian Ministry of Health (LB and FP), the Italian
Ministry of Scientific Research, MIUR, PRIN and FIRB (LB), INSERM l’Association pour la
Recherche sur le Cancer, ARC n°3283 (JJF) and a HMR-GIP Aventis fellowship (to M.P. )
Address correspondence: Dr. Luca Battistini, Neuroimmunology Unit,
Santa Lucia Foundation Via Ardeatina 306-354, 00179 Rome, Italy;
Phone: 0039-0651501521, Fax: 0039-0651501553, Email: [email protected];
Counts: 3599 words (abstract 158 words), 24436 characters, 26 pages, 3 figures;
1720 words (537 figure legends + 1183 references)
Scientific heading: Immunobiology
page 1
Copyright (c) 2004 American Society of Hematology
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Abstract
Upon recognition of nonpeptidic phosphoantigens, human Vδ2 T lymphocytes enter a lineage
differentiation pattern that determines the generation of memory cells with a range of effector
functions. Here, we show that within the effector memory Vδ2 population, two distinct and
complementary subsets with regard to phenotype, mode of activation and type of responses can
be identified: Vδ2 TEMh cells, which express high levels of chemokine receptors, but low
levels of perforin and of NK receptors (NKRs) and which produce large amounts of IFN-γ and
TNF-α in response to TCR-specific stimulation by phosphoantigens; and Vδ2 TEMRA cells,
which constitutively express several NKRs, high amounts of perforin, but low levels of
chemokine receptors and of IFN-γ. These NK-like cells are refractory to phosphoantigen but
respond to activation via FcγRIII (CD16) and are highly active against tumoral target cells.
Thus, circulating Vδ2 T lymphocytes comprise two functionally diverse subsets of effector
memory cells which may be discriminated on the basis of CD16 expression.
page 2
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Introduction
Human Vδ2-TCR+ lymphocytes constitute an unconventional lymphoid population. In
striking contrast to “conventional” lymphocytes bearing the αβ TCR, Vδ2 T lymphocytes
exhibit innate reactivity to MHC-unrestricted microbial and tumoral non-peptide antigens
which leads to proliferation, release of Th1 cytokines and perforin-mediated killing.1-3 Most
circulating Vδ2 cells are central memory and effector memory T cells,4,5 reflecting selective
activation by common environmental antigens, such as phosphorylated metabolites. Expression
of cell surface receptors for chemokines6 and for MHC class I molecules7,8 on a large fraction
of Vδ2 lymphocytes is also consistent with their memory phenotype.
Circulating naive αβ T cells use CD62L and CCR7 to bind high endothelial venules and
to migrate to lymph nodes, respectively.9 They also express the CD45RA isoform and the
costimulatory receptor CD27, but after primary antigen encounter surface expression of these
markers is gradually switched off.10,11 On the other hand, experienced T cells comprise central
memory (CD45RA-, CCR7+), effector memory (CD45RA-, CCR7-), and CD45RA+ effector
memory cells (CD45RA+, CCR7-), which respectively produce increasing amounts of
perforin.9,12-15 Likewise, while the majority of Vγ9Vδ2 T lymphocytes from cord blood are
naive (CD45RA+, and CD27+), most of their mature counterparts in adult blood from healthy
individuals have lost the CD45RA receptor.5 In addition, the effector functions of mature
circulating Vδ2 T cells comprise a potent cytolytic activity mediated by the release of
perforin,16 which is not produced by cord blood γδ T cells.17 CD45RA- CD27+ cells are
abundant in lymph nodes and lack immediate effector functions. Conversely, memory
CD45RA-CD27- and CD45RA+CD27- effector cells are poorly represented in lymph nodes
but home in inflamed tissue and display immediate effector functions.18
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Recent evidences have revealed that NK cells can be divided in two distinct
subpopulations with unique effector attributes based on expression of CD16.19 This receptor
binds IgG complexed to antigens and mediates phagocytosis, signal transduction and antibodydependent cellular cytotoxicity. Given the striking similarities between NK and γδ cells, we
investigated whether a similar distinction applied also for the latter population. Indeed, by
multiparameter phenotyping of blood Vδ2 T lymphocytes at the single cell level using
polychromatic flow cytometry, we define and characterize two different effector memory Vδ2
T cell subsets on the basis of CD16 expression. We describe their distinct phenotypes, patterns
of stimulation and functional responses. Finally, we suggest that these cells represent two
different pathways of maturation for circulating γδ T cells which are discriminated by CD16
expression.
Materials and Methods
Cell preparation and culture
Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated by
Ficoll-Hypaque gradient centrifugation (Pharmacia, Uppsala, Sweden) and cultured at 106
cells/ml in RPMI 1640 supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 2mM
L-glutamine, 20mM HEPES, and 10U/ml penicillin and streptomycin (Life Technologies,
Grand Island, NY).
Freshly collected PBMC comprising 1-3 % of TCRVγ9Vδ2+ cells were cultured in bulk (106
cells/ml) in complete culture medium supplemented with phosphoantigen (BrHPP 200 nM,
Innate Pharma, Marseilles) and IL-2 (100-400 U/ml, Sanofi-Synthélabo, Labège). for 2-4
weeks as described.20 On average, the cell lines comprised >95% TCRVγ9Vδ2+ after 3 weeks
of culture, and were used without further separation procedure in the specified analysis.
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Highly purified Vδ2 TEMh and Vδ2 TEMRA cell subsets were sorted by polychromatic
flow-cytometry on the MoFlo (DakoCytomation). Vδ2 T cell clones were obtained by single
cell sorting, and maintained in culture as previously described.21
Intracellular staining
Freshly isolated PBMC were stimulated with 10 nM BrHPP and treated with brefeldin
A (10 µM, Sigma-Aldrich). After 6 hours of stimulation, cells were stained following standard
procedures and analysed on the MoFlo cytometer. Results are expressed as % of Vδ2+ IFNγ+ T
cells on total Vδ2 T lymphocytes.
Flow cytometry
7-color FACS analysis was performed on a DakoCytomation MoFlo cytometer, and 5color FACS analysis was performed on a Coulter F-500 cytometer. Data was compensated and
analyzed using FlowJo software (TreeStar, USA). In one set of experiments data was collected
and acquired using RXP software (Coulter).
PBMC were isolated from blood samples
according to standard procedures. 1 x 106 cells were used for each staining. Monoclonal
antibodies (conjugated with the appropriate fluorochrome) were added at previously defined
optimal concentrations. The following antibodies were used: CD3 ECD®, CD62L ECD®,
CD27 PC5®, CD16 PE-Cy7, NKG2A PE, CD158.1 PE, CD158.2 PE, CD45RA ECD® and
Streptavidin PE-Cy7 from Beckman Coulter (USA); Vδ2 FITC and PE, biotinilated Vδ2,
CD45RA FITC, PE and APC, CD94 FITC, NKAT2 FITC, CCR6 PE, CCR5 FITC, CXCR3
FITC, CD16 PE-Cy7 and CyChrome®, Perforin FITC, IFN-γ APC from BD Pharmingen (San
Diego, CA, USA); CD27 FITC and CD62L APC-Cy7 from Caltag.
For functional studies cells were sorted at high speed (>30.000 cells/sec) on the MoFlo.
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PhosphoERK immunoblots
Highly purified (>95%) Vδ2+ T cells and clones were stimulated with 20 nM BrHpp at
37º C at four time points (5, 15, 30 and 60 minutes). Stimulation through the Fc receptor was
achieved by staining cells with purified α-CD16 (Pharmingen) for 30 min on ice, and after
washing off unbound Ab, cross-linking with GAM (Southern Biotechnologies) (1.5 µg/106
cells) for 5 min at 37º C. Following stimulation samples were lysed in SDS sample buffer.
Analysis of protein components was performed on 10% polyacrylamide gels, loading the same
volume of total lysate for each experimental condition. Proteins were transferred to PVDF
membranes (Amersham Biosciences) and blots were probed overnight at 4°C with antiphospho-p44/42 MAP Kinase (Cell Signaling), followed by horseradish peroxidase-coupled
secondary antibody (Cell Signaling). Samples were analysed using ECL Plus Western Blotting
Detection Reagent (Amersham Biosciences). Quantification was performed by Kodak Image
Station (KDS IS440CF 1.1).
Extracellular cytokine release
Vδ2 T cell clones were plated at 1x105 cells/well and stimulated with Brhpp 100 nM or
plate bound with purified anti-CD16 antibody (10 µg/ml), anti-HLAI (10 µg/ml) in the
presence of IL-2 50 U/ml (Roche Diagnostic). The samples were maintained at 37 °C for 24
and then supernatants were harvested. The presence of IFNγ was determined by a standard
two-site sandwich ELISA. Abs for IFNγ (purchased from Pharmingen). Enhanced proteinbinding ELISA plates (Nunc Maxisorb; Nunc Maxi Corp., Roskilde, Denmark) were used.
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Polychromatic synaptic transfer analysis
The Daudi lymphoma and HT29 colon carcinoma cell targets were labelled by PKH67
(Sigma-Aldrich) as described,22,23 rinsed and added at a 5-to-1 cell ratio to whole PBMC
(600,000 cells per well). The two groups were mixed together in complete RPMI culture
medium, pelleted by centrifugation (1 min., 700 rpm) and left for one hour at 37°C in CO2
incubator for synaptic transfer. Cells were subsequently dissociated by washing with PBS
containing 0.5 mM EDTA, and PBMC subsets were revealed by staining for 20 min. at 4°C
with the following mAb mix: TCRVδ2-PE-Cy7, CD16-Cy, CD27-APC, CD62L-ECD,
CD45RA-PE. Using a polychromatic cytometric (MoFlo) analysis, after gating out of the
PKH67bright tumoral targets, the following subsets were gated: Vδ2+ non-effectors (TCRVδ2+,
CD62L+, CD27+, CD16-), Vδ2 TEMh (TCRVδ2+, CD62L-, CD27-, CD45RA-, CD16-) and Vδ2
TEMRA (TCRVδ2+, CD62L-, CD27-, CD45RA+, CD16+). Synaptic transfer on each subset was
then analyzed by comparing the mean fluorescence intensity for labelling by PKH67 at t0 min.
and t60 min. of co-culture.22,23
Cytotoxicity assay
Highly purified (> 95%) NK cells, Vδ2 TEMRA cells and Vδ2 TEMh cells were sorted
by High Speed Cell Sorting (MoFlo, DakoCytomation) from freshly isolated PBMCs. These
cell subsets were used for cytotoxicity assays, as previously described,24 using Daudi cells as
target cells. Briefly: target cells were labelled with 250nM carboxyfluorescein diacetate
succinimydyl ester (CFSE purchased from Molecular Probes). The effector population were
seeded with a constant number of CFSE-labelled daudi cells (100.000) at different E:T ratios
(from 10:1 to 0.3:1), and incubated for 4 hours in a 5% CO2 atmosphere at 37°C. In parallel
target cells were incubated alone to measure basal apoptosis. Cell mixture were then labelled
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with 20 µg/ml 7-aminoactinomycin D (7-AAD purchased from Sigma-Aldrich) and analysed
by flow cytometry.
Results
Phenotypic and functional definition of VJ9VG2 lymphocyte subsets
Effector memory subsets of human Vγ9Vδ2 T lymphocytes in the blood from healthy
donors were defined by direct staining of freshly collected PBMC with a panel of fluorophoreconjugated monoclonal antibodies and polychromatic flow cytometry. The panel comprised
antibodies directed against TCRVδ2, CD27, CD62L, CD45RA and CD16. All blood samples
comprised four discrete Vδ2 subpopulations defined by the following phenotypes:18 naive
(Vδ2 TN): TCRVδ2+ CD62L+ CD27+ CD45RA+ CD16-; central memory (Vδ2 TCM): TCRVδ2+
CD62L+ CD27+ CD45RA- CD16-; effector memory (Vδ2 TEMh): TCRVδ2+ CD62L- CD27CD45RA- CD16-; and (CD45)RA+ effector memory (Vδ2 TEMRA): TCRVδ2+ CD62L- CD27CD45RA+ CD16+ (Fig. 1A). While the Vδ2 TN and Vδ2 TCM correspond to non-effector
cells,5,18 the Vδ2 TEMh and Vδ2 TEMRA subsets display immediate effector functions. A variable
percentage of CD27+CD62L- cells was found in all donors tested. This subset likely represents
an intermediate stage between central memory cells and effector cells, as CD62L is rapidly
downregulated upon antigenic stimulation, while CD27 is downregulated at a slower rate (data
not shown). PBMC cultured for 6 hours with the phosphoantigen BrHPP were labelled for
intracellular perforin and IFNγ in addition to the 5 surface-markers previously described. As
shown in Figure 1B, within Vδ2+ T cells, several subpopulations could be identified based on
expression of perforin and production of IFN-γ: non-effectors, (perforin-IFN-γ-) and effectors
that produce either perforin or IFN-γ. Progressive gating on each group for multiparametric
analysis of surface marker expression confirmed that both TCRVδ2+ CD62L+ CD27+
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CD45RA+ CD16- and TCRVδ2+ CD62L+ CD27+ CD45RA- CD16- subsets are non-effectors
(not shown). By contrast, the TCRVδ2+ CD62L- CD27- CD45RA- CD16- cells were perforinlow
and exploited IFN-γhigh responses, while the TCRVδ2+ CD62L- CD27- CD45RA+ CD16+ cells
were perforinhigh but did not produce IFN-γ; these subsets were referred to as Vδ2 TEMh and
Vδ2 TEMRA, respectively (Fig.1B). Healthy individuals (n=26) presented highly variable
frequencies of both subsets (1-40x10-4 Vδ2 TEMh and 3-300x10-4 Vδ2 TEMRA, Fig. 1C) while
Vδ2 T cell lines maintained in vitro with phosphoantigen and IL2 prominently comprised Vδ2
TEMh cells (80% Vδ2 TEMh and 0-4% Vδ2 TEMRA, Fig. 1C). Thus by phenotypic and functional
analogy with differentiation stages of αβ T lymphocytes,25 circulating human Vδ2 T
lymphocytes comprise variable frequencies of naive and central memory non-effector cells,
together with two effector memory subsets composed of helpers and cytotoxic cells.
Differential expression of NK and chemokine receptors on VG2 T cell clones
The co-existence of two distinct effector subsets of memory Vδ2 T lymphocytes was
confirmed by flow cytometry on isolated clones in culture. Vδ2 TEMh are perforinlow and do not
express NK-receptors such as CD16, CD158 and NKAT2, express low levels of NKG2A,
CD94, but high levels of the chemokine receptors CXCR3, CCR5 and CCR6, thereby
resembling conventional Th1 cells.6,26 On the contrary, Vδ2 TEMRA cells display an almost
complementary phenotypic pattern, including high levels of perforin, several NK-receptors
(CD16, CD94, NKG2A, CD158, NKAT2), but low levels of chemokine receptors (CXCR3,
CCR5 and CCR6; Fig. 2A). Thus, the Vδ2 TEMRA phenotype depicts cytotoxic NK-like γδ T
cells. Moreover, to assess induction or modulation of these receptors we stimulated Vδ2 cell
clones with phosphoantigen and we measured the expression levels. Vδ2+ TEMh clones
downregulated chemokine receptor expression in response to phosphoantigens, as shown
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previously,27 whereas the Vδ2+ TEMRA cells showed a stable chemokine expression confirming
the unresponsiveness of this subset of γδ T cells to stimulation through the TCR
(supplementary Figure 1).
TEMRA and TEMh have distinct patterns of activation and responses
Occasionally, flow cytometric analysis on freshly isolated PBMC from healthy donors reveals
the presence of two subsets of Vδ2 T cells with distinct levels of TCR expression.
Multiparametric analysis performed on such sample revealed that TCR Vδ2high T cells are Vδ2
TEMh cells (CD45RA-CD16-), whereas the TCR Vδ2low T cells are Vδ2 TEMRA cells
(CD45RA+CD16+) (Fig. 2B). TCR Vδ2low (TEMRA) and Vδ2high (TEMh) from this donor were
sorted, and functional comparison confirmed that these two subsets have distinct patterns of
activation and responses (Fig. 2C). In agreement with their higher surface expression of the
phosphoantigen-specific TCR Vδ2, a stronger ERK signalling is induced in Vδ2 TEMh than in
Vδ2 TEMRA cells by BrHPP. Although still responding weakly to the phosphoantigen, Vδ2
TEMRA lymphocytes strongly induce their ERK signalling pathway upon the NK-like activation
through FcγIII-R ligation with anti-CD16 antibody (Fig. 2C). Level of expression of the Vδ2
TCR was stable in culture in both Vδ2+ TEMRA and Vδ2+ TEMh, and the differences in ERK1/2
phosphorylation were even more striking compared to those observed on freshly isolated Vδ2+
cells (supplementary Fig. 2)
Accordingly, cytometric analysis from donors with large numbers of Vδ2 TEMRA (see
representative sample in Fig.3A) showed that intracellular production of IFN-γ (and of TNF-α,
data not shown) is induced by BrHPP stimulation in Vδ2 TEMh but not in Vδ2 TEMRA cells,
whereas conversely Vδ2 TEMRA are able to produce IFN-γ following triggering through CD16.
These data obtained ex vivo from PBMC by intracellular staining were confirmed using Vδ2 T
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cell clones of Vδ2 TCM, Vδ2 TEMh or Vδ2 TEMRA in cultures stimulated with BrHPP:
phosphoantigen induced the release of IFN-γ by Vδ2 TEMh cells only (Fig3B). In agreement
with results obtained with PBMC (Fig.3A), Vδ2 TEMRA cells do not produce IFN-γ in response
to BrHPP but do so following cross-linking of CD16 (Fig.3B). Furthermore, we compared the
cytotoxic activity of the two different subsets of Vδ2 effector cells by challenging Vδ2 TEMh,
Vδ2 TEMRA and NK cell clones with a tumoral cell target (Daudi). The results showed a strong
lytic activity exerted by the NK cells (up to 90%), an “intermediate” lythic activity of Vδ2
TEMRA cells (up to 40%), and no cytotoxic effect by Vδ2 TEMh cells. It is of note that in these
experiments we analyzed spontaneous cytotoxic activity of the lymphocyte subsets, while
generally γδ T cell cytotoxicity is measured followed cellular activation, and is in any case not
as vigorous as we describe here.
Engagement of target cells
To confirm at the single cell level the cytotoxic activity of the different subsets of
effector Vδ2 lymphocytes, we took advantage of their ability to engage cell targets prior to
lethal hit delivery. Since engagement of target cells through the immunological synapses of
Vδ2 lymphocytes,20 of NK cells22 and of CD8 αβ T lymphocytes23 leads to synaptic transfer,
we used this assay with bulk PBMC to compare at the single cell level the ability of Vδ2 TEMh
and Vδ2 TEMRA cells to engage cell targets. Vδ2 TEMh appear to engage Daudi cells (Fig. 3D),
but to a similar extent as the non-effector Vδ2 TN and Vδ2 TCM counterparts (not shown). Of
all Vδ2 PBMC subsets, the Vδ2 TEMRA subset is the most active in establishing synapses with
cancer cell targets such as the Daudi Burkitt lymphoma cell line (Fig. 3D) or with HT29
colorectal cancer (data not shown).
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Together, these features uncover two distinct subsets of effector/memory Vδ2 T cells with
different antigen recognition pathways, phenotype and functional capabilities.
Discussion
Vδ2 T cells constitute an abundant reservoir of antitumoral and anti-infectious
effectors. The specific and straightforward targeting of Vγ9Vδ2 T lymphocytes by
phosphoantigens, alkylamines and aminobiphosphonates makes these cells particularly
attractive for anti-cancer immunotherapies28 or anti-infectious vaccines.29 Thus, it is crucial to
define precisely how specific activation by synthetic phosphoantigens may be exploited to tune
this response. Here we show that, similar to αβ T lymphocytes, functional heterogeneity of
memory cells also applies to the Vδ2 T cell subset. Expression of CD27 and CD45RA,
migratory routes and effector functions define four successive differentiation steps for Vδ2 T
cells.18 The complete phenotypic and functional characterization of these effector memory
subsets by polychromatic flow cytometry discloses differential responses to phosphoantigens.
Vδ2 human effector memory T cells comprise TEMh and TEMRA cells. The former, composed of
TCR-specific, phosphoantigen-responsive helper cells is over-represented in phosphoantigendriven cell lines and clones in culture and corresponds to the most extensively studied Vδ2 T
cells.
On the other hand are the formerly described highly cytotoxic NK-like γδ T
lymphocytes,21,30,31 characterized further in this study as phosphoantigen-unresponsive Vδ2
TEMRA cells and corresponding to the CD16+ γδ lymphocytes depicted elsewhere.32
By analogy with CD8 αβ TEMRA cells15,33 and considering the shorter telomeres of Vδ2
TEM cells,18 it is conceivable that Vδ2 TEMRA cells correspond to a late stage of Vδ2
differentiation. Strongly suggestive of a progressive selection for the fittest effectors,25
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differentiation into Vδ2 TEMRA produces the most highly active antitumoral effectors among
Vδ2 T cells. Given the negative influence of phosphoantigens on the in vitro induction of Vδ2
TEMRA cells from whole PBMC demonstrated by this study, their presence in vivo most
probably reflects a prolonged absence of stimulation by microbial phosphoantigens, as
proposed for αβ TEMRA cells.33 Indeed, the pool of proliferating non-effector γδ cells must
generate either TCR-dependent helpers or NK-like cytolytic effectors by some as yet
incompletely defined terminal maturation switch. While phosphoantigen clearly drives
proliferation of precursor pools18 and maturation of Vδ2 TEMh (as seen within cultured Vδ2 T
cell lines in Fig. 1C and for Vδ2 PBMC, Fig.3A), the antigen-independent generation of Vδ2
TEMRA cells remains enigmatic. Several reports have described appearance of αβ CD8 TEMRA
lymphocytes following viral infections.14,34 Likewise, the frequency ex vivo of Vδ2 TEMRA cells
is usually very low in healthy donors18 and often appears elevated in several pathological
conditions (our unpublished observations). However, some healthy donors having a basal Vδ2
T cell number higher than 5% of total PBMC may express a larger fraction of Vδ2 TEMRA
probably as the consequence of subclinical infections or exposure to environmental pathogens.
These Vδ2 TEMRA cells were shown to represent the main Vδ2 T cell subset present in ascite
and cerebrospinal fluids of tuberculosis patients,18 confirming the migratory capability of these
end-stage effectors.
Other non-peptide antigens than phosphoantigens, which selectively activate Vδ2 T
lymphocytes, have been described, including therapeutic aminobisphosphonates35 and
alkylamines derived from microbes and edible plants.36 However, these compounds appear
unlikely triggers for Vδ2 TEMRA cells, since specificity for these ligands has been convincingly
assigned to reactive Vδ2 TCR rather than to expression of other cell surface receptors such as
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CD16. In addition, recent clinical investigations have documented Th1-type in vivo response of
human Vδ2 lymphocytes to aminobisphosphonates,29,37,38 and to an alkylamine-rich diet.39
On the other hand, we have occasionally found individuals whose Vδ2 T cells express
different levels of cell surface TCR. It is well established that surface expression of the TCR
correlates to the developmental and activation status of the cell, and that its regulation affects T
cells function.39 Following encounter with antigen, the TCR is quickly internalised from the
cell surface,40-43 and depending on the strength of the stimulation, T cells can become
unresponsive to subsequent antigenic challenges. Indeed, while Vδ2high cells were responsive
to stimulation with BrHPP, Vδ2low cells displayed low reactivity. The striking phenotypic
differences between Vδ2high and Vδ2low cells tempt us to suggest that the latter are the result of
a vigorous antigenic stimulation ex-vivo, maybe following an infection, which determines TCR
downregulation, acquisition of NK markers and the progression to the final steps of the
differentiation to effectors. This scenario is similar to that of CD8+ αβ T cells, which have
been shown to re-express the CD45RA isoform when terminally differentiated,14 and to
upregulate NKR expression.44 Effector cells are thought to have a short life-span, while central
memory cells represent the pool of long-lived cells from which effectors are generated at the
time of need.25 In contrast, terminally differentiated Vδ2 TEMRA cells appear to persist in vivo
for extended periods of time (Angelini D.F. et al., manuscript in preparation), and have been
reported to be the most represented γδ T cells in inflamed tissues.18
Thus, given i) their marked ability to engage and to lyse tumoral cell targets, ii) their
high content of perforin, and iii) their ability to secrete TNF-α and IFN-γ upon CD16 (but not
phosphoantigen-) mediated activation, the terminally differentiated Vδ2 TEMRA cells represent
a distinct and critical pool of cytotoxic effectors within the γδ T cell population.
Our results have demonstrated that, as suggested for NK cells,19 expression of CD16
could discriminate the two subsets of Vδ2 T lymphocytes with different functional roles. The
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CD16 negative subset has the ability to produce high levels of cytokines, expresses low levels
of KIRs and is poorly cytotoxic, while the CD16 positive subset presents high levels of KIRs
and is a potent cytotoxic effector cell. Therefore, we suggest that Vδ2+ T cells should not be
considered as a homogeneous population, but rather as a combination of distinct effector
subsets. Moreover the similarity with NK cells is again underlined by the important role of
cytokinic environment (rather than antigen recognition) in the development of distinct
subsets.19
Future studies will now aim at elucidating the conditions which favor the selective
generation of human Vδ2 TEMRA lymphocytes, due to the need of generating cytotoxic effectors
for anticancer immunotherapy purposes.
SUPPLEMENTAL MATERIAL IS AVAILABLE ONLINE AT THE TIME OF FINAL
PUBLICATION ONLY.
Acknowledgments: We acknowledge Innate Pharma (Marseille) for providing BrHPP and
Sanofi-Synthélabo (Labège) for generous supply of IL2.
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Figure legends
Figure 1: Polychromatic profiling of the VG2 T cell subsets
A: 5-colours flow cytometry discriminates between different subsets of naïve and memory Vδ2
T cells from human PBMC. Sequential gating using morphology and TCR expression defines
non-effector and effector Vδ2+ cell subsets according to CD27 and CD62L expression, which
are respectively sub-divided according to expression of CD45RA and CD16. B: After 6 hrs of
culture of PBMC with phosphoantigen, intracellular staining for perforin and IFNγ followed by
7-colour flow cytometry as above defines two functionally distinct subsets of effector memory
Vδ2 T cells by showing that Vδ2TEMh are IFNγ-producing lymphocytes with the
TCRVδ2+CD27-CD62L-CD45RA-CD16- phenotype, while Vδ2TEMRA are perforin-producing
lymphocytes with the TCRVδ2+CD27-CD62L-CD45RA+CD16+ phenotype. C: frequency of
Vδ2TEMh and Vδ2TEMRA cells in PBMC from four representative healthy donors and two
cultured cell lines.
Figure 2: Phenotype and activation patterns of VG2TEMh and VG2TEMRA lymphocytes
A: NKR and chemokine receptors of representative TEMh (upper panels) and TEMRA (lower
panels) TCR Vδ2+ clones. Cultured Vδ2 T cell clones are characterized as Vδ2TEMh on the
basis of their CD16-CD45RA-perforinlow phenotype, or as Vδ2TEMRA when they are
CD16+CD45RA+perforinhigh (MFI for intracellular perforin are given).
B: Vδ2TEMh and Vδ2TEMRA PBMC freshly drawn from a healthy donor with different levels of
cell surface TCR Vδ2. C: PhosphoERK immunoblots of lysates from the above cells (5 x 105
cells /point). After sorting using gates shown in B , in vitro activation of the collected cells and
production of their lysates, the p-Erk immunoblots reveal a higher response to BrHPP in
page 22
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Vδ2TEMh than in Vδ2TEMRA, which respond better to stimulation anti-CD16. The data is
representative of two independent experiment on different donors.
Figure 3: Different functional responses of VG2TEMh and VG2TEMRA lymphocytes
A: Cytometric panels refer to a representative healthy donor. Left panel shows cells gated for
Vδ2 expressing double staining for CD16 and perforin, indicating that both CD16+perforin+
and CD16-perforin- effector cells were equally present in the chosen individual. Freshly
isolated PBMC where stimulated with BrHPP or anti-CD16 for 6 hours and intracellular
staining for IFN-γ was performed (right panels).
B: Vδ2 TCM, TEMh and TEMRA T cell clones were obtained as previously described.28 The cells
were then stimulated with BrHpp (100 nM) or with anti-CD16 (10 µg/ml). Supernatants were
harvested after 24 hours and IFN-γ was measured by ELISA. Anti HLAI was used as control.
The data is representative of three independent ELISA experiment on different donors.
C: NK cell, Vδ2 TEMh and TEMRA T cell subsets, freshly isolated from a healthy donor, were
used for cytotoxic assay against Daudi cells. The graph shows a strong lytic activity exerted by
the NK cells (up to 90%), an “intermediate” lytic activity of Vδ2 TEMRA cells (up to 40% ), and
no cytotoxic effect by Vδ2 TEMh cells. The data is representative of two independent
experiment.
D: Synaptic transfer by Vδ2TEMh and Vδ2TEMRA lymphocytes from bulk PBMC co-incubated
for one hour with PKH67-labelled Daudi target cells. Using FL1 for PKH67 detection,
PKH67+ Daudi cells were gated out, the Vδ2 cell subsets were defined by 5-colour flow
cytometry as in Fig. 1 after simultaneous labeling for surface TCRVδ2, CD27, CD62L,
CD45RA and CD16. Numbers give the PKH67 mfi of the specified cells prior to and after coincubation.
page 23
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page 24
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page 25
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page 26
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Prepublished online June 3, 2004;
doi:10.1182/blood-2004-01-0331
FcγRIII discriminates between two subsets of Vγ9Vδ2 effector cells with
different responses and activation pathways
Daniela F ANGELINI, Giovanna BORSELLINO, Mary POUPOT, Adamo DIAMANTINI, Remy
POUPOT, Giorgio BERNARDI, Fabrizio POCCIA, Jean-Jacques FOURNIE and Luca BATTISTINI
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