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Blood, 15 September 2004, Vol. 104, No. 6, pp. 1801-1807. Prepublished online as a Blood First Edition Paper on June 3, 2004; DOI 10.1182/blood-2004-01-0331. This Article Abstract Full Text (PDF) Supplemental Figures All Versions of this Article: 2004-01-0331v1 104/6/1801    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Angelini, D. F. Articles by Battistini, L. Search for Related Content PubMed PubMed Citation Articles by Angelini, D. F. Articles by Battistini, L. Related Collections Immunobiology Signal Transduction Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  IMMUNOBIOLOGY FcRIII discriminates between 2 subsets of V9V2 effector cells with different responses and activation pathways Daniela F. Angelini, Giovanna Borsellino, Mary Poupot, Adamo Diamantini, Rémy Poupot, Giorgio Bernardi, Fabrizio Poccia, Jean-Jacques Fournié, and Luca Battistini From the Neuroimmunology Unit, Santa Lucia Foundation, Scientific Institute (IRCCS), Rome, Italy; the Départment Oncogénèse et Signalisation dans les Cellules Hématopoiétiques, Unité 563 de l'Institut National de la Santé etdela Recherche Médicale, Centre de Physiopathologie de Toulouse Purpan, Toulouse, France; the Department of Neuroscience, University of Rome "Tor Vergata," and the Laboratory of Immunopathology, Padiglione Del Vecchio, National Institute for Infectious Diseases, Rome, Italy.    Abstract Top Abstract Introduction Materials and methods Results Discussion References   Upon recognition of nonpeptidic phosphoantigens, human V2 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 V2 population, 2 distinct and complementary subsets with regard to phenotype, mode of activation, and type of responses can be identified: V2 TEMh cells, which express high levels of chemokine receptors, but low levels of perforin and of natural killer receptors (NKRs) and which produce large amounts of interferon (IFN-) and tumor necrosis factor (TNF-) in response to T-cell receptor (TCR)–specific stimulation by phosphoantigens; and V2TEMRA cells, which constitutively express several NKRs, high amounts of perforin, but low levels of chemokine receptors and of IFN-. These NK-like cells are refractory to phosphoantigen but respond to activation via FcRIII (CD16) and are highly active against tumoral target cells. Thus, circulating V2T lymphocytes comprise 2 functionally diverse subsets of effector memory cells that may be discriminated on the basis of CD16 expression.    Introduction Top Abstract Introduction Materials and methods Results Discussion References   Human V2–T-cell receptor (TCR)+ lymphocytes constitute an unconventional lymphoid population. In striking contrast to "conventional" lymphocytes bearing the TCR, V2 T lymphocytes exhibit innate reactivity to major histocompatibility complex (MHC)–unrestricted microbial and tumoral nonpeptide antigens, which leads to proliferation, release of T-helper 1 (Th1) cytokines, and perforin-mediated killing.1-3 Most circulating V2 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 V2 lymphocytes is also consistent with their memory phenotype. Circulating naive 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, whereas most V9V2 T lymphocytes from cord blood are naive (CD45RA+, 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 V2 T cells comprise a potent cytolytic activity mediated by the release of perforin,16 which is not produced by cord blood 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 Recent evidence has revealed that natural killer (NK) cells can be divided in 2 distinct subpopulations with unique effector attributes based on expression of CD16.19 This receptor binds immunoglobulin G (IgG) complexes to antigens and mediates phagocytosis, signal transduction, and antibody-dependent cellular cytotoxicity. Given the striking similarities between NK and cells, we investigated whether a similar distinction applied also for the latter population. Indeed, by multiparameter phenotyping of blood V2 T lymphocytes at the single-cell level using polychromatic flow cytometry, we define and characterize 2 different effector memory V2 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 2 different pathways of maturation for circulating T cells, which are discriminated by CD16 expression.    Materials and methods Top Abstract Introduction Materials and methods Results Discussion References   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% (vol/vol) heat-inactivated fetal bovine serum, 2 mM L-glutamine, 20 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], and 10 U/mL penicillin and streptomycin (Life Technologies, Grand Island, NY). Freshly collected PBMCs comprising 1% to 3% of TCRV9V2+ cells were cultured in bulk (106 cells/mL) in complete culture medium supplemented with phosphoantigen (BrHPP; 200 nM; Innate Pharma, Marseilles, France) and interleukin 2 (IL-2; 100-400 U/mL; Sanofi-Synthélabo, Labège, France) for 2 to 4 weeks as described.20 On average, the cell lines comprised more than 95% TCRV9V2+ after 3 weeks of culture, and were used without further separation procedure in the specified analysis. Highly purified V2 TEMh and V2 TEMRA cell subsets were sorted by polychromatic flow cytometry on the MoFlo (DakoCytomation, Glostrup, Denmark). V2 T-cell clones were obtained by single-cell sorting, and maintained in culture as previously described.21 Intracellular staining Freshly isolated PBMCs were stimulated with 10 nM BrHPP and treated with brefeldin A (10 µM; Sigma-Aldrich, St Louis, MO). After 6 hours of stimulation, cells were stained following standard procedures and analyzed on the MoFlo cytometer. Results are expressed as percent of V2+ IFN+ T cells on total V2 T lymphocytes. Flow cytometry Seven-color fluorescence activated cell sorting (FACS) analysis was performed on a DakoCytomation MoFlo cytometer, and 5-color FACS analysis was performed on a Coulter F-500 cytometer. Data were compensated and analyzed using FlowJo software (TreeStar, Ashland, OR). In one set of experiments data were collected and acquired using RXP software (Beckman, Coulter, Fullerton, CA). PBMCs were isolated from blood samples according to standard procedures. For each staining 1 x 106 cells were used. 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 phycoerythrin (PE)–Cy7, NKG2A PE, CD158.1 PE, CD158.2 PE, CD45RA ECD, and streptavidin PE-Cy7 from Beckman Coulter; V2 fluorescein isothiocyanate (FITC) and PE, biotinylated V2, CD45RA FITC, PE, and allophycocyanin (APC), CD94 FITC, NKAT2 FITC, CCR6 PE, CCR5 FITC, CXCR3 FITC, CD16 PE-Cy7, and CyChrome, perforin FITC, and interferon- (IFN-) APC from BD Pharmingen (San Diego, CA); CD27 FITC and CD62L APC-Cy7 from Caltag (Burlingame, CA). For functional studies cells were sorted at high speed (> 30 000 cells/second) on the MoFlo. PhosphoERK immunoblots Highly purified (> 95%) V2+ T cells and clones were stimulated with 20 nM BrHPP at 37° C at 4 time points (5, 15, 30, and 60 minutes). Stimulation through the Fc receptor was achieved by staining cells with purified -CD16 (Pharmingen) for 30 minutes on ice, and after washing off unbound antibody (Ab), cross-linking with goat anitmouse (GAM; Southern Biotechnologies, Birmingham, AL; 1.5 µg/106 cells) for 5 minutes at 37° C. Following stimulation samples were lysed in sodium dodecyl sulfate (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 polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences, Piscataway, NJ) and blots were probed overnight at 4° C with antiphospho-p44/42 mitogen activated protein (MAP) kinase (Cell Signaling, Beverly, MA), followed by horseradish peroxidase–coupled secondary antibody (Cell Signaling). Samples were analyzed using ECL Plus Western Blotting Detection Reagent (Amersham Biosciences). Quantification was performed by Kodak Image Station (KDS IS440CF 1.1; Kodak, Rochester, NY). Extracellular cytokine release V2 T-cell clones were plated at 1 x 105 cells/well and stimulated with 100 nM BrHPP or plate bound with purified anti-CD16 antibody (10 µg/mL), anti–HLA-I (10 µg/mL) in the presence of 50 U/mL IL-2 (Roche Diagnostic, Milan, Italy). The samples were maintained at 37° C for 24 hours and then supernatants were harvested. The presence of IFN- was determined by a standard 2-site sandwich enzyme-linked immunosorbent assay (ELISA). Abs for IFN- were purchased from Pharmingen. Enhanced protein-binding ELISA plates (Nunc Maxisorb; Nunc Maxi, Roskilde, Denmark) were used. Polychromatic synaptic transfer analysis The Daudi lymphoma and HT29 colon carcinoma cell targets were labeled by PKH67 (Sigma-Aldrich) as described,22,23 rinsed, and added at a 5:1 cell ratio to whole PBMCs (600 000 cells per well). The 2 groups were mixed together in complete RPMI culture medium, pelleted by centrifugation (1 minute, 150 g) and left for 1 hour at 37° C in CO2 incubator for synaptic transfer. Cells were subsequently dissociated by washing with phosphate-buffered saline (PBS) containing 0.5 mM EDTA [ethylenediaminetetraacetic acid], and PBMC subsets were revealed by staining for 20 minutes at 4° C with the following monoclonal antibody (mAb) mix: TCRV2-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: V2+ noneffectors (TCRV2+, CD62L+, CD27+, CD16–), V2TEMh (TCRV2+, CD62L–, CD27–, CD45RA–, CD16–) and V2 TEMRA (TCRV2+, CD62L–, CD27–, CD45RA+, CD16+). Synaptic transfer on each subset was then analyzed by comparing the mean fluorescence intensity for labeling by PKH67 at time 0 minutes and time 60 minutes of coculture.22,23 Cytotoxicity assay Highly purified (> 95%) NK cells, V2 TEMRA cells, and V2 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 labeled with 250 nM carboxyfluorescein diacetate succinimydyl ester (CFSE; Molecular Probes, Eugene, OR). The effector population were seeded with a constant number of CFSE-labeled Daudi cells (100 000) at different effector-to-target (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 was then labeled with 20 µg/mL 7-aminoactinomycin D (7-AAD; Sigma-Aldrich) and analyzed by flow cytometry.    Results Top Abstract Introduction Materials and methods Results Discussion References   Phenotypic and functional definition of V9V2 lymphocyte subsets Effector memory subsets of human V9V2 T lymphocytes in the blood from healthy donors were defined by direct staining of freshly collected PBMCs with a panel of fluorophore-conjugated monoclonal antibodies and polychromatic flow cytometry. The panel comprised antibodies directed against TCRV2, CD27, CD62L, CD45RA, and CD16. All blood samples comprised 4 discrete V2 subpopulations defined by the following pheno-types:18 naive (V2 TN): TCRV2+ CD62L+ CD27+ CD45RA+ CD16–; central memory (V2 TCM): TCRV2+ CD62L+ CD27+ CD45RA– CD16–; effector memory (V2 TEMh): TCRV2+ CD62L– CD27–CD45RA– CD16–; and (CD45)RA+ effector memory (V2 TEMRA): TCRV2+ CD62L– CD27–CD45RA+ CD16+ (Figure 1A). While the V2TN and V2TCM correspond to noneffector cells,5,18 the V2 TEMh and V2 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 down-regulated upon antigenic stimulation, whereas CD27 is down-regulated at a slower rate (data not shown). PBMCs cultured for 6 hours with the phosphoantigen BrHPP were labeled for intracellular perforin and IFN- in addition to the 5 surface markers previously described. As shown in Figure 1B, within V2+ T cells, several subpopulations could be identified based on expression of perforin and production of IFN-: noneffectors, (perforin–IFN-–) and effectors that produce either perforin or IFN-. Progressive gating on each group for multiparametric analysis of surface marker expression confirmed that both TCRV2+ CD62L+ CD27+ CD45RA+ CD16– and TCRV2+ CD62L+ CD27+ CD45RA– CD16– subsets are noneffectors (not shown). By contrast, the TCRV2+ CD62L– CD27–CD45RA– CD16– cells were perforinlow and exploited IFN-high responses, whereas the TCRV2+ CD62L– CD27–CD45RA+ CD16+ cells were perforinhigh but did not produce IFN-; these subsets were referred to as V2TEMh and V2 TEMRA, respectively (Figure 1B). Healthy individuals (n = 26) presented highly variable frequencies of both subsets (1-40 x 10–4 V2 TEMh and 3-300 x 10–4 V2 TEMRA; Figure 1C) while V2 T-cell lines maintained in vitro with phosphoantigen and IL-2 prominently comprised V2 TEMh cells (80% V2 TEMh and 0-4% V2 TEMRA; Figure 1C). Thus, by phenotypic and functional analogy with differentiation stages of T lymphocytes,25 circulating human V2 T lymphocytes comprise variable frequencies of naive and central memory noneffector cells, together with 2 effector memory subsets composed of helpers and cytotoxic cells. View larger version (50K): [in this window] [in a new window]   Figure 1.. Polychromatic profiling of the V2 T-cell subsets. (A) 5-color flow cytometry discriminates between different subsets of naive and memory V2 T cells from human PBMCs. Sequential gating using morphology and TCR expression defines noneffector and effector V2+ cell subsets according to CD27 and CD62L expression, which are respectively subdivided according to expression of CD45RA and CD16. (B) After 6 hours of culture of PBMCs with phosphoantigen, intracellular staining for perforin and IFN- followed by 7-color flow cytometry as described in "Materials and methods" defines 2 functionally distinct subsets of effector memory V2 T cells by showing that V2TEMh are IFN-–producing lymphocytes with the TCRV2+CD27– CD62L–CD45RA– CD16– phenotype, whereas V2TEMRA are perforin-producing lymphocytes with the TCRV2+CD27– CD62L–CD45RA+CD16+ phenotype. (C) Frequency of V2TEMh and V2TEMRA cells in PBMCs from 4 representative healthy donors and 2 cultured cell lines.   Differential expression of NK and chemokine receptors on V2 T-cell clones The coexistence of 2 distinct effector subsets of memory V2 T lymphocytes was confirmed by flow cytometry on isolated clones in culture. V2 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, V2 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; Figure 2A). Thus, the V2 TEMRA phenotype depicts cytotoxic NK-like T cells. Moreover, to assess induction or modulation of these receptors we stimulated V2 cell clones with phosphoantigen and we measured the expression levels. V2+ TEMh clones down-regulated chemokine receptor expression in response to phosphoantigens, as shown previously,27 whereas the V2+ TEMRA cells showed a stable chemokine expression confirming the unresponsiveness of this subset of T cells to stimulation through the TCR (Figure S1; see the Supplemental Figures link at the top of the online article on the Blood website). View larger version (53K): [in this window] [in a new window]   Figure 2.. Phenotype and activation patterns of V2TEMh and V2TEMRA lymphocytes. (A) NKR and chemokine receptors of representative TEMh (top panels) and TEMRA (bottom panels) TCR V2+ clones. Cultured V2 T-cell clones are characterized as V2TEMh on the basis of their CD16–CD45RA– perforinlow pheno-type, or as V2TEMRA when they are CD16+CD45RA+ perforinhigh (mean fluorescence intensity [MFI] for intracellular perforin is given). (B) V2TEMh and V2TEMRA PBMCs freshly drawn from a healthy donor with different levels of cell surface TCR V2. (C) PhosphoERK immunoblots of lysates from V2high and V2low cells (5 x 105 cells/point). After sorting using gates shown in panel B, in vitro activation of the collected cells, and production of their lysates, the p-Erk immunoblots reveal a higher response to BrHPP in V2TEMh than in V2TEMRA, which respond better to stimulation with anti-CD16. The data are representative of 2 independent experiments on different donors.   TEMRA and TEMh have distinct patterns of activation and responses Occasionally, flow cytometric analysis on freshly isolated PBMCs from healthy donors reveals the presence of 2 subsets of V2 T cells with distinct levels of TCR expression. Multiparametric analysis performed on such samples revealed that TCR V2high T cells are V2 TEMh cells (CD45RA–CD16–), whereas the TCR V2low T cells are V2TEMRA cells (CD45RA+CD16+; Figure 2B). TCR V2low (TEMRA) and V2high (TEMh) from this donor were sorted, and functional comparison confirmed that these 2 subsets have distinct patterns of activation and responses (Figure 2C). In agreement with their higher surface expression of the phosphoantigen-specific TCR V2, a stronger ERK signaling is induced in V2 TEMh than in V2TEMRA cells by BrHPP. Although still responding weakly to the phosphoantigen, V2 TEMRA lymphocytes strongly induce their ERK signaling pathway upon the NK-like activation through FcIII-R ligation with anti-CD16 antibody (Figure 2C). Level of expression of the V2 TCR was stable in culture in both V2+ TEMRA and V2+ TEMh, and the differences in ERK1/2 phosphorylation were even more striking compared with those observed on freshly isolated V2+ cells (Figure S2). Accordingly, cytometric analysis from donors with large numbers of V2TEMRA (Figure 3A, representative sample) showed that intracellular production of IFN- (and of TNF-, data not shown) is induced by BrHPP stimulation in V2TEMh but not in V2TEMRA cells, whereas conversely V2 TEMRA are able to produce IFN- following triggering through CD16. These data obtained ex vivo from PBMCs by intracellular staining were confirmed using V2 T-cell clones of V2 TCM, V2 TEMh, or V2 TEMRA in cultures stimulated with BrHPP: phosphoantigen induced the release of IFN- by V2 TEMh cells only (Figure 3B). In agreement with results obtained with PBMCs (Figure 3A), V2TEMRA cells do not produce IFN- in response to BrHPP but do so following cross-linking of CD16 (Figure 3B). Furthermore, we compared the cytotoxic activity of the 2 different subsets of V2 effector cells by challenging V2 TEMh, V2 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" lytic activity of V2 TEMRA cells (up to 40%), and no cytotoxic effect by V2TEMh cells. It is of note that in these experiments we analyzed spontaneous cytotoxic activity of the lymphocyte subsets, while generally T-cell cytotoxicity is measured following cellular activation, and is in any case not as vigorous as we describe here. View larger version (34K): [in this window] [in a new window]   Figure 3.. Different functional responses of V2TEMh and V2TEMRA lymphocytes. (A) Cytometric panels refer to a representative healthy donor. Left panel shows cells gated for V2 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 PBMCs were stimulated with BrHPP or anti-CD16 for 6 hours and intracellular staining for IFN- was performed (right panels). (B) V2TCM,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- was measured by ELISA. Anti-HLAI was used as control. The data are representative of 3 independent ELISA experiments on different donors. Data represent means ± SD. (C) NK cell, V2TEMh, 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 V2TEMRA cells (up to 40%), and no cytotoxic effect by V2TEMh cells. The data are representative of 2 independent experiments. (D) Synaptic transfer by V2TEMh and V2TEMRA lymphocytes from bulk PBMCs coincubated for 1 hour with PKH67-labeled Daudi target cells. Using FL1 for PKH67 detection, PKH67+ Daudi cells were gated out, the V2 cell subsets were defined by 5-color flow cytometry as in Figure 1, after simultaneous labeling for surface TCRV2, CD27, CD62L, CD45RA, and CD16. Numbers give the PKH67 MFI of the specified cells prior to and after coincubation.   Engagement of target cells To confirm at the single-cell level the cytotoxic activity of the different subsets of effector V2 lymphocytes, we took advantage of their ability to engage cell targets prior to lethal hit delivery. Since engagement of target cells through the immunologic synapses of V2 lymphocytes,20 of NK cells22 and of CD8 T lymphocytes23 leads to synaptic transfer, we used this assay with bulk PBMCs to compare at the single-cell level the ability of V2 TEMh and V2 TEMRA cells to engage cell targets. V2 TEMh appear to engage Daudi cells (Figure 3D), but to a similar extent as the noneffector V2 TN and V2 TCM counterparts (not shown). Of all V2 PBMC subsets, the V2 TEMRA subset is the most active in establishing synapses with cancer cell targets such as the Daudi Burkitt lymphoma cell line (Figure 3D) or with HT29 colorectal cancer (data not shown). Together, these features uncover 2 distinct subsets of effector/memory V2 T cells with different antigen recognition pathways, phenotype, and functional capabilities.    Discussion Top Abstract Introduction Materials and methods Results Discussion References   V2 T cells constitute an abundant reservoir of antitumoral and anti-infectious effectors. The specific and straightforward targeting of V9V2 T lymphocytes by phosphoantigens, alkylamines, and aminobiphosphonates makes these cells particularly attractive for anticancer 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 T lymphocytes, functional heterogeneity of memory cells also applies to the V2 T-cell subset. Expression of CD27 and CD45RA, migratory routes, and effector functions define 4 successive differentiation steps for V2 T cells.18 The complete phenotypic and functional characterization of these effector memory subsets by polychromatic flow cytometry discloses differential responses to phosphoantigens. V2 human effector memory T cells comprise TEMh and TEMRA cells. The former, composed of TCR-specific, phosphoantigen-responsive helper cells is overrepresented in phosphoantigen-driven cell lines and clones in culture and corresponds to the most extensively studied V2 T cells. On the other hand are the formerly described highly cytotoxic NK-like T lymphocytes,21,30,31 characterized further in this study as phosphoantigen-unresponsive V2 TEMRA cells and corresponding to the CD16+ lymphocytes depicted elsewhere.32 By analogy with CD8 TEMRA cells15,33 and considering the shorter telomeres of V2 TEM cells,18 it is conceivable that V2 TEMRA cells correspond to a late stage of V2 differentiation. Strongly suggestive of a progressive selection for the fittest effectors,25 differentiation into V2 TEMRA produces the most highly active antitumoral effectors among V2 T cells. Given the negative influence of phosphoantigens on the in vitro induction of V2 TEMRA cells from whole PBMCs demonstrated by this study, their presence in vivo most probably reflects a prolonged absence of stimulation by microbial phosphoantigens, as proposed for TEMRA cells.33 Indeed, the pool of proliferating noneffector 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 V2 TEMh (as seen within cultured V2 T-cell lines in Figure 1C and for V2 PBMCs; Figure 3A), the antigen-independent generation of V2 TEMRA cells remains enigmatic. Several reports have described appearance of CD8 TEMRA lymphocytes following viral infections.14,34 Likewise, the frequency ex vivo of V2TEMRA cells is usually very low in healthy donors18 and often appears elevated in several pathologic conditions (D.F.A. et al, unpublished observations, December 2003). However, some healthy donors having a basal V2 T-cell number higher than 5% of total PBMCs may express a larger fraction of V2 TEMRA probably as the consequence of subclinical infections or exposure to environmental pathogens. These V2 TEMRA cells were shown to represent the main V2 T-cell subset present in ascite and cerebrospinal fluids of tuberculosis patients,18 confirming the migratory capability of these end-stage effectors. Other nonpeptide antigens than phosphoantigens, which selectively activate V2 T lymphocytes, have been described, including therapeutic aminobisphosphonates35 and alkylamines derived from microbes and edible plants.36 However, these compounds appear unlikely triggers for V2 TEMRA cells, since specificity for these ligands has been convincingly assigned to reactive V2 TCR rather than to expression of other cell-surface receptors such as CD16. In addition, recent clinical investigations have documented the Th1-type in vivo response of human V2 lymphocytes to aminobisphosphonates,29,37,38 and to an alkylamine-rich diet.39 On the other hand, we have occasionally found individuals whose V2 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-cell function.39 Following encounter with antigen, the TCR is quickly internalized 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 V2high cells were responsive to stimulation with BrHPP, V2low cells displayed low reactivity. The striking phenotypic differences between V2high and V2low 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 down-regulation, acquisition of NK markers, and the progression to the final steps of the differentiation to effectors. This scenario is similar to that of CD8+ T cells, which have been shown to re-express the CD45RA isoform when terminally differentiated,14 and to up-regulate NKR expression.44 Effector cells are thought to have a short life span, whereas 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 V2 TEMRA cells appear to persist in vivo for extended periods of time (D.F.A. et al, manuscript in preparation), and have been reported to be the most represented T cells in inflamed tissues.18 Thus, given (1) their marked ability to engage and to lyse tumoral cell targets, (2) their high content of perforin, and (3) their ability to secrete TNF- and IFN- upon CD16-mediated (but not phosphoantigen-mediated) activation, the terminally differentiated V2 TEMRA cells represent a distinct and critical pool of cytotoxic effectors within the T-cell population. Our results have demonstrated that, as suggested for NK cells,19 expression of CD16 could discriminate the 2 subsets of V2 T lymphocytes with different functional roles. The CD16– subset has the ability to produce high levels of cytokines, expresses low levels of killer inhibitory receptors (KIRs) and is poorly cytotoxic, while the CD16+ subset presents high levels of KIRs and is a potent cytotoxic effector cell. Therefore, we suggest that V2+ 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 that favor the selective generation of human V2 TEMRA lymphocytes, due to the need of generating cytotoxic effectors for anticancer immunotherapy purposes.    Acknowledgements   We acknowledge Innate Pharma (Marseille, France) for providing BrHPP, and Sanofi-Synthélabo (Labège, France) for generous supply of IL-2.    Footnotes   Submitted January 28, 2004; accepted May 3, 2004. Prepublished online as Blood First Edition Paper, June 3, 2004; DOI 10.1182/blood-2004-01-0331. Supported by institutional grants from the Italian Ministry of Health (L.B., F.P.), the Italian Ministry of Scientific Research, the Ministero dell'Instruzione, dell'Universitè e della Ricerca (MIUR), the Progetti di Ricerca di Interesse Nazionale (PRIN), and the Fondo per gli Investimenti della Ricerca di Base (FIRB) (L.B.), INSERM l'Association pour la Recherche sur le Cancer, Association pour la Recherche sur le Cancer (ARC) no. 3283 (J.J.F.), and an Hoechst Marion Roussel–Groupment d'Interêt Public (HMR-GIP) Aventis fellowship (M.P.). D.F.A. and G.B. contributed equally to this work. The online version of the article contains a data supplement. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Luca Battistini, Neuroimmunology Unit, Santa Lucia Foundation Via Ardeatina 306-354, 00179 Rome, Italy; e-mail: l.battistini{at}hsantalucia.it.    References Top Abstract Introduction Materials and methods Results Discussion References   Bonneville M, Fournié JJ. Gamma Delta Lymphocytes. Encyclopedia of Life Sciences (Nature Publishing Group). 2001:1-7. Available at http://www.els.net/doi:10.1038/npg.els.0001195. Accessed January 2004. Hayday AC. cells: a right time and a right place for a conserved third way of protection. Ann Rev Immunol 2000;18: 975-1026.[CrossRef][Medline] [Order article via Infotrieve] Bendelac A, Bonneville M, Kearney JF. Autoreactivity by design: innate B and T lymphocytes. Nat Rev Immunol. 2001;1: 177-186.[CrossRef][Medline] [Order article via Infotrieve] Parker CM, Groh V, Band H, et al. Evidence for extrathymic changes in the T cell receptor gamma/delta repertoire. 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