Human gamma delta T cells: Evolution and ligand recognition.

The γδ T cell lineage in humans remains much of an enigma due to the low number of defined antigens, the non-canonical ways in which these cells respond to their environment and difficulty in tracking this population in vivo. In this review, we survey a comparative evolutionary analysis of the primate V, D and J gene segments and contrast these findings with recent progress in defining antigen recognition by different populations of γδ T cells in humans. Signatures of both purifying and diversifying selection at the Vδ and Vγ gene loci are placed into context of Vδ1+ γδ T cell recognition of CD1d presenting different lipids, and Vγ 9Vδ2 T cell modulation by pyrophosphate-based phosphoantigens through the butyrophilins BTN3A. From this comparison, it is clear that co-evolution between γδ TCRs and these ligands is likely occurring, but the diversity inherent in these recombined receptors is an important feature in ligand surveillance.

γδ T cell activation by bispecific antibodies.

Bispecific antibodies have been successfully introduced into clinical application. γδ T cells are of special interest for tumor immunotherapy, due to their recognition of pyrophosphates that are overproduced by many tumor cells resulting in HLA-nonrestricted tumor cell killing. Here we describe in detail a [(Her2)2 × Vγ9] tribody construct that targets human Vγ9 T cells to HER2-expressing tumor cells. The direct comparison with other selective Vγ9 T cell agonists including phosphoantigens and nitrogen-containing bisphosphonates revealed the superiority of the [(Her2)2 × Vγ9] tribody in triggering γδ T cell-mediated tumor cell killing with negligible induction of γδ T cell death. In contrast, phosphoantigens and bisphosphonates are potent inducers of γδ T cell proliferation but less efficient enhancers of γδ T cell-mediated tumor cell killing. Collectively, our data identify unique properties of a γδ T cell-targeting [(Her2)2 × Vγ9] tribody which make it an attractive candidate for clinical application in γδ T cell-based tumor immunotherapy.

Human γδ T-cell responses in infection and immunotherapy: common mechanisms, common mediators?

Upon receiving the Nobel Prize in Physiology or Medicine in 1987, Susumu Tonegawa referred to the then recent discovery of the γδ T-cell receptor and stated that "while the function of the T cells bearing this receptor is currently unknown (…) these T cells may be involved in an entirely new aspect of immunity". [Tonegawa, S., Scand. J. Immunol. 1993. 38: 303-319]. Twenty-five years of intense research later this ambivalent view still holds true. Immunologists now appreciate that γδ T cells indeed represent a highly intriguing "new aspect of immunity" that is unique and distinct from conventional lymphocytes, yet even scientists in the field still struggle to understand the molecular basis of γδ T-cell responses, especially with respect to the enigmatic mode of antigen recognition. Here, we portray the peculiar responsiveness of human Vγ9/Vδ2 T cells to microorganisms, tumor cells and aminobisphosphonates, in an attempt to integrate the corresponding - and at times confusing - findings into a "theory of everything" that may help explain how such diverse stimuli result in similar γδ T-cell responses via the recognition of soluble low molecular weight phosphoantigens.

Expression and function of PD-1 in human γδ T cells that recognize phosphoantigens.

Programmed cell death-1 (PD-1) is an inhibitory receptor and plays an important role in the regulation of αß T cells. Little is known, however, about the role of PD-1 in γδ T cells. In this study, we investigated the expression and function of PD-1 in human γδ T cells. Expression of PD-1 was rapidly induced in primary γδ T cells following antigenic stimulation, and the PD-1(+) γδ T cells produced IL-2. When PD-1(+) γδ T cells were stimulated with Daudi cells with and without programmed cell death ligand-1 (PD-L1) expression, the levels of IFN-γ production and cytotoxicity in response to PD-L1(+) Daudi cells were diminished compared to the levels seen in response to PD-L1(-) Daudi cells. The attenuated effector functions were reversed by anti-PD-L1 mAb. When PD-1(+) γδ T cells were challenged by PD-L1(+) tumors pretreated with zoledronate (Zol), which induced γδ TCR-mediated signaling, the resulting reduction in cytokine production was only slight to moderate compared to the reduction seen when PD-1(+) γδ T cells were challenged by PD-L1(-) tumors. In addition, cytotoxic activity of PD-1(+) γδ T cells against Zol-treated PD-L1(+) tumors was comparable to that against Zol-treated PD-L1(-) tumors. These results suggest that TCR triggering may partially overcome the inhibitory effect of PD-1 in γδ T cells.

Monocytes and gammadelta T cells: close encounters in microbial infection.

gammadelta T cells comprise an evolutionarily conserved yet poorly understood subset of T cells. Numerous features place these unconventional lymphocytes at the branching point between antigen-presenting cells and natural killer cells of the innate immune system and major-histocompatibility-complex-restricted alphabeta T cells of the adaptive immune system. We propose a role for human Vgamma9/Vdelta2 T cells in the generation of monocyte-derived inflammatory dendritic cells during infection. Our model incorporates the peculiar innate-like specificity of Vgamma9/Vdelta2 T cells for the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), co-recruitment of monocytes and Vgamma9/Vdelta2 T cells to sites of infection, and their crosstalk, with profound consequences for the initiation of antigen-specific alphabeta T-cell responses. Vgamma9/Vdelta2 T cells act thus as a cellular switch between innate and adaptive defence mechanisms.

Phosphoantigens overcome human TCRVgamma9+ gammadelta Cell immunosuppression by TGF-beta: relevance for cancer immunotherapy.

Human gammadelta cells expressing TCRVgamma9 are HLA-unrestricted CTLs with high relevance for cancer immunotherapy. Many tumor cell types produce TGF-beta, however, a cytokine strongly immunosuppressive for conventional T CD4, CD8, and NK cells. Whether TGF-beta also inhibits TCRVgamma9+ lymphocytes was unknown. Because phosphoantigens (PAgs), such as bromohydrin pyrophosphate, selectively activate the antitumor functions of TCRVgamma9+ T cells, in this study, we investigated whether TGF-beta modulates these functions. We report that TGF-beta does not block activation of TCRVgamma9+ T cells but inhibits their PAg/IL-2-induced proliferation and maturation into effector cells and finally reduces the cytotoxic activity of these gammadelta T cells when exposed to lymphoma target cells. TGF-beta did not bias their differentiation pattern toward gammadelta Th17 or gammadelta regulatory T cells. Nevertheless, increasing doses of PAg stimulus countered TGF-beta inhibition. So, although TGF-beta impairs TCRVgamma9+ gammadelta cells like other cytolytic lymphocytes, PAg alone or combined to therapeutic mAb has the ability to bypass its immunosuppressive activity.

Vgamma2Vdelta2 T Cell Receptor recognition of prenyl pyrophosphates is dependent on all CDRs.

gammadelta T cells differ from alphabeta T cells in the Ags they recognize and their functions in immunity. Although most alphabeta TCRs recognize peptides presented by MHC class I or II, human gammadelta T cells expressing Vgamma2Vdelta2 TCRs recognize nonpeptide prenyl pyrophosphates. To define the molecular basis for this recognition, the effect of mutations in the TCR CDR was assessed. Mutations in all CDR loops altered recognition and cover a large footprint. Unlike murine gammadelta TCR recognition of the MHC class Ib T22 protein, there was no CDR3delta motif required for recognition because only one residue is required. Instead, the length and sequence of CDR3gamma was key. Although a prenyl pyrophosphate-binding site was defined by Lys109 in Jgamma1.2 and Arg51 in CDR2delta, the area outlined by critical mutations is much larger. These results show that prenyl pyrophosphate recognition is primarily by germline-encoded regions of the gammadelta TCR, allowing a high proportion of Vgamma2Vdelta2 TCRs to respond. This underscores its parallels to innate immune receptors. Our results also provide strong evidence for the existence of an Ag-presenting molecule for prenyl pyrophosphates.

γδ T-APCs: a novel tool for immunotherapy?

The series of seminal articles in this book clearly illustrate the multi-functional nature of γδ T cells. Some of the functions correlate with the tissue tropism of distinct γδ T cell subsets whereas others appear to result from oligoclonal selection. Here, we discuss the antigen-presenting cell (APC) function of the major subset of circulating γδ T cells, Vγ9/Vδ2 T cells, present in human blood. During tissue culture, Vγ9/Vδ2 T cells uniformly respond to a class of non-peptide antigens, so-called prenyl pyrophosphates, derived from stressed host cells or from microbes. It is this feature that distinguishes human (and primate) Vγ9/Vδ2 T cells from αß and γδ T cells of all other species and that forms the basis for detailed studies of human Vγ9/Vδ2 T cells. One of the consequences of Vγ9/Vδ2 T cell activation is the rapid acquisition of APC characteristics (γδ T-APCs) reminiscent of mature dendritic cells (DCs). In the following discussion, we will discriminate between the potential use of γδ T-APCs as a cellular vaccine in immunotherapy and their role in anti-microbial immunity. Exploiting the APC function in γδ T-APCs represents a true novelty in current immunotherapy research and may lead to effective, anti-tumor immunity in cancer patients.

Bromohydrin pyrophosphate enhances antibody-dependent cell-mediated cytotoxicity induced by therapeutic antibodies.

In human blood, 1% to 5% of lymphocytes are gammadelta T cells; they mostly express the gammadelta T-cell receptor (TCR)Vgamma9, recognize nonpeptide phosphoantigens (PAgs) produced by microbes and tumor cells, and mediate different modes of lytic activities directed against tumor target cells. Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by cytolytic lymphoid cells is essential for the clinical activity of anticancer monoclonal antibodies (mAbs), but whether PAgs affect ADCC by gammadelta T cells is unknown. Here we report that, in association with the CD20(+)-specific mAb rituximab (RTX), the synthetic PAg bromohydrin pyrophosphate (BrHPP) increased TCRVgamma9(+) cell binding to CD20(+) lymphoma cells in vitro. This combination activated phospho-ZAP70 and phospho-ERK1/2 signaling in TCRVgamma9(+) cells and strongly enhanced their ADCC activity. We obtained similar results with BrHPP in the context of the mAbs alemtuzumab and trastuzumab. Furthermore, BrHPP enhanced RTX-mediated depletion of CD20(+) cells in vitro from peripheral blood mononuclear cells of healthy subjects and enhanced ADCC by gammadelta T cells from patients with chronic lymphocytic leukemia. In cynomolgus macaques, a regimen combining RTX, BrHPP, and IL2 activated TCRVgamma9(+) lymphocytes and enhanced B-cell depletion from blood and lymph nodes. Thus, the combination with BrHPP PAg is able to improve the efficacy of cancer immunotherapy by therapeutic mAbs.

Tuberculosis-induced variant IL-4 mRNA encodes a cytokine functioning as growth factor for (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate-specific Vgamma2Vdelta2 T cells.

The possibility that mycobacterial infections induce variant cytokine mRNA encoding a functionally distinct protein for immune regulation has not been addressed. In this study, we reported that Mycobacterium tuberculosis and bacillus Calmette-Guérin infections of macaques induced expression of variant IL-4 (VIL-4) mRNA encoding a protein comprised of N-terminal 97 aa identical with IL-4, and unique C-terminal 96 aa including a signaling-related proline-rich motif. While VIL-4 could be stably produced as intact protein, the purified VIL-4 induced apparent expansion of phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP)-specific Vgamma2Vdelta2 T cells in dose- and time-dependent manners. The unique C-terminal 96 aa bearing the proline-rich motif (PPPCPP) of VIL-4 appeared to confer the ability to expand Vgamma2Vdelta2 T cells, since simultaneously produced IL-4 had only a subtle effect on these gammadelta T cells. Moreover, VIL-4 seemed to use IL-4R alpha for signaling and activation, as the VIL-4-induced expansion of Vgamma2Vdelta2 T cells was blocked by anti-IL-4R alpha mAb but not anti-IL-4 mAb. Surprisingly, VIL-4-expanded Vgamma2Vdelta2 T cells after HMBPP stimulation appeared to be heterologous effector cells capable of producing IL-4, IFN-gamma, and TNF-alpha. Thus, mycobacterial infections of macaques induced variant mRNA encoding VIL-4 that functions as growth factor promoting expansion of HMBPP-specific Vgamma2Vdelta2 T effector cells.

(E)-4-hydroxy-3-methyl-but-2 enyl pyrophosphate-stimulated Vgamma9Vdelta2 T cells possess T helper type 1-promoting adjuvant activity for human monocyte-derived dendritic cells.

Vgamma9Vdelta2 T cells respond to pyrophosphate antigens and display potent antitumour activity in vitro. We have investigated the potential of the most potent phosphoantigen known to activate Vgamma9Vdelta2 T cells, (E)-4-hydroxy-3-methyl-but-2 enyl pyrophosphate (HMB-PP), as an adjuvant for dendritic cell (DC)-based vaccines. A single stimulation of peripheral blood mononuclear cells with HMB-PP and IL-2 was sufficient to generate lines of effector memory Vgamma9Vdelta2 T cells that retained their cytolytic and cytokine secretion activities. These cells induced differentiation of DC into semi-mature antigen-presenting cells expressing CD86, CD11c, CD54, HLA-DR, CD83 and CD40, which secreted low levels of bioactive IL-12 but no IL-10. Vgamma9Vdelta2 T cells also strongly costimulated IL-12 release but inhibited IL-10 production by lipopolysaccharide (LPS)-stimulated DC. When substituted for Vgamma9Vdelta2 T cells, IFN-gamma did not induce full DC maturation but it augmented IL-12 and inhibited IL-10 release by LPS-stimulated DC, in a manner similar to HMB-PP-activated Vgamma9Vdelta2 T cells. Our findings indicate that Vgamma9Vdelta2 T cells, stimulated with nanomolar concentrations of HMB-PP, strongly promote T helper type 1 (Th1) responses through their ability to induce DC maturation and IL-12 secretion. This adjuvant activity may prove useful in DC-based cancer therapies.

Preferential Th1 cytokine profile of phosphoantigen-stimulated human Vγ9Vδ2 T cells.

Human Vγ9Vδ2 T cells recognise pyrophosphate-based antigens (phosphoantigens) and have multiple functions in innate and adaptive immunity, including a unique ability to activate other cells of the immune system. We used flow cytometry and ELISA to define the early cytokine profiles of Vγ9Vδ2 T cells stimulated in vitro with isopentenyl pyrophosphate (IPP) and (E)-4-hydroxy-3-methyl-but-2 enyl pyrophosphate (HMB-PP) in the absence and presence of IL-2 and IL-15. We show that fresh Vγ9Vδ2 T cells produce interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) within 4 hours of stimulation with phosphoantigen, but neither IL-10, IL-13, nor IL-17 was detectable up to 72 hours under these conditions. Cytokine production was not influenced by expression or lack, thereof, of CD4 or CD8. Addition of IL-2 or IL-15 caused expansion of IFN-γ-producing Vγ9Vδ2 T cells, but did not enhance IFN-γ secretion after 24-72 hours. Thus, phosphoantigen-stimulated Vγ9Vδ2 T cells have potential as Th1-biasing adjuvants for immunotherapy.

Regulation of Human γδ T Cells by BTN3A1 Protein Stability and ATP-Binding Cassette Transporters.

Activation of human Vγ9/Vδ2 T cells by "phosphoantigens" (pAg), the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) and the endogenous isoprenoid intermediate isopentenyl pyrophosphate, requires expression of butyrophilin BTN3A molecules by presenting cells. However, the precise mechanism of activation of Vγ9/Vδ2 T cells by BTN3A molecules remains elusive. It is not clear what conformation of the three BTN3A isoforms transmits activation signals nor how externally delivered pAg accesses the cytosolic B30.2 domain of BTN3A1. To approach these problems, we studied two HLA haplo-identical HeLa cell lines, termed HeLa-L and HeLa-M, which showed marked differences in pAg-dependent stimulation of Vγ9/Vδ2 T cells. Levels of IFN-γ secretion by Vγ9/Vδ2 T cells were profoundly increased by pAg loading, or by binding of the pan-BTN3A specific agonist antibody CD277 20.1, in HeLa-M compared to HeLa-L cells. IL-2 production from a murine hybridoma T cell line expressing human Vγ9/Vδ2 T cell receptor (TCR) transgenes confirmed that the differential responsiveness to HeLa-L and HeLa-M was TCR dependent. By tissue typing, both HeLa lines were shown to be genetically identical and full-length transcripts of the three BTN3A isoforms were detected in equal abundance with no sequence variation. Expression of BTN3A and interacting molecules, such as periplakin or RhoB, did not account for the functional variation between HeLa-L and HeLa-M cells. Instead, the data implicate a checkpoint controlling BTN3A1 stability and protein trafficking, acting at an early time point in its maturation. In addition, plasma membrane profiling was used to identify proteins upregulated in HMB-PP-treated HeLa-M. ABCG2, a member of the ATP-binding cassette (ABC) transporter family was the most significant candidate, which crucially showed reduced expression in HeLa-L. Expression of a subset of ABC transporters, including ABCA1 and ABCG1, correlated with efficiency of T cell activation by cytokine secretion, although direct evidence of a functional role was not obtained by knockdown experiments. Our findings indicate a link between members of the ABC protein superfamily and the BTN3A-dependent activation of γδ T cells by endogenous and exogenous pAg.

Vgamma9Vdelta2 T cells recovered from eyes of patients with Behçet's disease recognize non-peptide prenyl pyrophosphate antigens.

The phenotype and antigen-specificity of T cells expanded by mitogenic stimulation from intra-ocular fluid (IOF) samples of affected eyes of six Behçet's disease (BD) patients, and seven patients with other uveitis entities, were determined. High numbers of gammadelta T cells, predominantly Vgamma9Vdelta2 T cells, were only detected in the IOF-derived TCL of three BD patients. Whereas no TCL responded to heat shock protein (HSP) 65 kDa, reactivity to isopentyl pyrophosphate (IPP) and related non-peptide prenyl pyrophosphates (PPP) was restricted to the gammadelta T cell containing TCL. Upon IPP stimulation, these TCL secreted IFN-gamma but no IL-4. By single-cell analysis of intracellular IFN-gamma production and CD69 expression the IOF-derived IPP-specific T cells were identified as CD4(-)CD8(-) gammadelta T cells. The data presented suggest the infiltration of PPP-specific Vgamma9Vdelta2 Th1-like cells into the eye of BD patients with uveitis.

Bromohydrin pyrophosphate-stimulated Vgamma9delta2 T cells expanded ex vivo from patients with poor-prognosis neuroblastoma lyse autologous primary tumor cells.

Gamma/delta T cells (Vgamma9delta2) contribute to innate immunity and exert natural cytotoxicity against a variety of tumors. Using a synthetic phosphoantigen (Bromohydrin Pyrophosphate, BrHPP), we amplified Vgamma9delta2 T cells in vitro from neuroblastoma patients. In the presence of BrHPP and low doses of IL-2, robust proliferation of Vgamma9delta2 T cells was obtained from peripheral blood mononuclear cells (PBMC) harvested at diagnosis. Moderate proliferation was observed from PBMC harvested after stem cell transplantation, whereas modest levels of Vgamma9delta2 T cells were obtained from PBMC harvested after induction therapy. Proliferation was observed after a single in vitro stimulation with BrHPP. After 21 days in culture, Vgamma9delta2 T cells represented more than 80% of cultured cells (a 50-fold expansion from baseline). Moreover, BrHPP-amplified Vgamma9delta2 T cells from patients-expressed activation markers and were able to lyse allogeneic and autologous neuroblasts. This cytotoxic activity was gammadelta T-cell receptor-dependent. Clinical trials using BrHPP are warranted in patients with poor-prognosis neuroblastoma, either to expand patient-derived Vgamma9delta2 T cells ex vivo or by direct administration to in vivo to boost the pool of resident Vgamma9delta2 T cells in vivo.

Highly active microbial phosphoantigen induces rapid yet sustained MEK/Erk- and PI-3K/Akt-mediated signal transduction in anti-tumor human gammadelta T-cells.

BACKGROUND: The unique responsiveness of Vgamma9Vdelta2 T-cells, the major gammadelta subset of human peripheral blood, to non-peptidic prenyl pyrophosphate antigens constitutes the basis of current gammadelta T-cell-based cancer immunotherapy strategies. However, the molecular mechanisms responsible for phosphoantigen-mediated activation of human gammadelta T-cells remain unclear. In particular, previous reports have described a very slow kinetics of activation of T-cell receptor (TCR)-associated signal transduction pathways by isopentenyl pyrophosphate and bromohydrin pyrophosphate, seemingly incompatible with direct binding of these antigens to the Vgamma9Vdelta2 TCR. Here we have studied the most potent natural phosphoantigen yet identified, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), produced by Eubacteria and Protozoa, and examined its gammadelta T-cell activation and anti-tumor properties. METHODOLOGY/PRINCIPAL FINDINGS: We have performed a comparative study between HMB-PP and the anti-CD3epsilon monoclonal antibody OKT3, used as a reference inducer of bona fide TCR signaling, and followed multiple cellular and molecular gammadelta T-cell activation events. We show that HMB-PP activates MEK/Erk and PI-3K/Akt pathways as rapidly as OKT3, and induces an almost identical transcriptional profile in Vgamma9(+) T-cells. Moreover, MEK/Erk and PI-3K/Akt activities are indispensable for the cellular effects of HMB-PP, including gammadelta T-cell activation, proliferation and anti-tumor cytotoxicity, which are also abolished upon antibody blockade of the Vgamma9(+) TCR Surprisingly, HMB-PP treatment does not induce down-modulation of surface TCR levels, and thereby sustains gammadelta T-cell activation upon re-stimulation. This ultimately translates in potent human gammadelta T-cell anti-tumor function both in vitro and in vivo upon transplantation of human leukemia cells into lymphopenic mice, CONCLUSIONS/SIGNIFICANCE: The development of efficient cancer immunotherapy strategies critically depends on our capacity to maximize anti-tumor effector T-cell responses. By characterizing the intracellular mechanisms of HMB-PP-mediated activation of the highly cytotoxic Vgamma9(+) T-cell subset, our data strongly support the usage of this microbial antigen in novel cancer clinical trials.

Nonpeptide antigens, presentation mechanisms, and immunological memory of human Vgamma2Vdelta2 T cells: discriminating friend from foe through the recognition of prenyl pyrophosphate antigens.

Human Vgamma2Vdelta2 T cells play important roles in mediating immunity against microbial pathogens and have potent anti-tumor activity. Vgamma2Vdelta2 T cells recognize the pyrophosphorylated isoprenoid intermediates (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate in the foreign 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, and isopentenyl pyrophosphate (IPP), an intermediate in the self-mevalonate pathway. Infection with bacteria and protozoa using the MEP pathway leads to the rapid expansion of Vgamma2Vdelta2 T cells to very high numbers through preferential recognition of HMBPP. Activated Vgamma2Vdelta2 T cells produce proinflammatory cytokines and chemokines, kill infected cells, secrete growth factors for epithelial cells, and present antigens to alphabeta T cells. Vgamma2Vdelta2 T cells can also recognize high levels of IPP in certain tumors and in cells treated with pharmacological agents, such as bisphosphonates and alkylamines, that block farnesyl pyrophosphate synthase. Activated Vgamma2Vdelta2 T cells are able to kill most tumor cells because of recognition by T-cell receptor and natural killer receptors. The ubiquitous nature of the antigens converts essentially all Vgamma2Vdelta2 T cells to memory cells at an early age. Thus, primary infections with HMBPP-producing bacteria are perceived by Vgamma2Vdelta2 T cells as a repeat infection. Extensive efforts are underway to harness these cells to treat a variety of cancers and to provide microbial immunity.

A tuberculosis vaccine based on phosphoantigens and fusion proteins induces distinct gammadelta and alphabeta T cell responses in primates.

Phosphoantigens are mycobacterial non-peptide antigens that might enhance the immunogenicity of current subunit candidate vaccines for tuberculosis. However, their testing requires monkeys, the only animal models suitable for gammadelta T cell responses to mycobacteria. Thus here, the immunogenicity of 6-kDa early secretory antigenic target-mycolyl transferase complex antigen 85B (ESAT-6-Ag85B) (H-1 hybrid) fusion protein associated or not to a synthetic phosphoantigen was compared by a prime-boost regimen of two groups of eight cynomolgus. Although phosphoantigen activated immediately a strong release of systemic Th1 cytokines (IL-2, IL-6, IFN-gamma, TNF-alpha), it further anergized blood gammadelta T lymphocytes selectively. By contrast, the hybrid H-1 induced only memory alphabeta T cell responses, regardless of phosphoantigen. These latter essentially comprised cytotoxic T lymphocytes specific for Ag85B (on average + 430 cells/million PBMC) and few IFN-gamma-secreting cells (+ 40 cells/million PBMC, equally specific for ESAT-6 and for Ag85B). Hence, in macaques, a prime-boost with the H-1/phosphoantigen subunit combination induces two waves of immune responses, successively by gammadelta T and alphabeta T lymphocytes.

Prolonged (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate-driven antimicrobial and cytotoxic responses of pulmonary and systemic Vgamma2Vdelta2 T cells in macaques.

Although phosphoantigen-specific Vgamma2Vdelta2 T cells appear to play a role in antimicrobial and anticancer immunity, mucosal immune responses and effector functions of these gammadelta T cells during infection or phospholigand treatment remain poorly characterized. In this study, we demonstrate that the microbial phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) plus IL-2 treatment of macaques induced a prolonged major expansion of circulating Vgamma2Vdelta2 T cells that expressed CD8 and produced cytotoxic perforin during their peak expansion. Interestingly, HMBPP-activated Vgamma2Vdelta2 T cells underwent an extraordinary pulmonary accumulation, which lasted for 3-4 mo, although circulating Vgamma2Vdelta2 T cells had returned to baseline levels weeks prior. The Vgamma2Vdelta2 T cells that accumulated in the lung following HMBPP/IL-2 cotreatment displayed an effector memory phenotype, as follows: CCR5+CCR7-CD45RA-CD27+ and were able to re-recognize phosphoantigen and produce copious amounts of IFN-gamma up to 15 wk after treatment. Furthermore, the capacity of massively expanded Vgamma2Vdelta2 T cells to produce cytokines in vivo coincided with an increase in numbers of CD4+ and CD8+ alphabeta T cells after HMBPP/IL-2 cotreatment as well as substantial perforin expression by CD3+Vgamma2- T cells. Thus, the prolonged HMBPP-driven antimicrobial and cytotoxic responses of pulmonary and systemic Vgamma2Vdelta2 T cells may confer immunotherapeutics against infectious diseases and cancers.