Vγ9Vδ2+ cells represent the major population of γδ T cells in primate blood and react in an MHC-unrestricted fashion to a set of low m.w. nonpeptide phosphoantigens. Two types of structurally related agonists have been discovered so far: the natural phosphoantigens (hydroxydimethyl allyl-pyrophosphate or isopentenyl-pyrophosphate (IPP)) acting directly on Vγ9Vδ2+ TCR and aminobisphosphonates, which block the mevalonate pathway in target cells, leading to accumulation of natural phosphoantigens that in turn activate Vγ9Vδ2+ cells. We demonstrate in the cynomolgus monkey that Vγ9Vδ2 can be manipulated in vivo with bromohydrin pyrophosphate (BrHPP)/Phosphostim, a potent synthetic agonist for which the mechanism of action is similar to natural phosphoantigens. Although of very short half-life, injection of BrHPP leads to strong activation of Vγ9Vδ2, inducing production of a high level of Th1 cytokines. Combination of BrHPP with low-dose rhIL-2 induces specific amplification of effector-memory peripheral Vγ9Vδ2 in blood in a dose-dependant manner. This transient response returns to baseline within 10–15 days. Successive infusions of BrHPP and rhIL-2 induce less vigorous expansions, suggesting a progressive exhaustion of the response. As no toxicity is detected with or without IL-2, this scheme represents a promising immunotherapeutic strategy for induction of systemic Th1 cytokines and massive expansion of γδ T cell subset with antitumor and anti-infectious properties.

Dominant Vγ2Vδ2 T-cell subset exist only in primates, and recognize phosphoantigen from selected pathogens including M. tuberculosis(Mtb). In vivo function of Vγ2Vδ2 T cells in tuberculosis remains unknown. We conducted mechanistic studies to determine whether earlier expansion/differentiation of Vγ2Vδ2 T cells during Mtb infection could increase immune resistance to tuberculosis in macaques. Phosphoantigen/IL-2 administration specifically induced major expansion and pulmonary trafficking/accumulation of phosphoantigen-specific Vγ2Vδ2 T cells, significantly reduced Mtb burdens and attenuated tuberculosis lesions in lung tissues compared to saline/BSA or IL-2 controls. Expanded Vγ2Vδ2 T cells differentiated into multifunctional effector subpopulations capable of producing anti-TB cytokines IFNγ, perforin and granulysin, and co-producing perforin/granulysin in lung tissue. Mechanistically, perforin/granulysin-producing Vγ2Vδ2 T cells limited intracellular Mtb growth, and macaque granulysin had Mtb-bactericidal effect, and inhibited intracellular Mtb in presence of perforin. Furthermore, phosphoantigen/IL2-expanded Vγ2Vδ2 T effector cells produced IL-12, and their expansion/differentiation led to enhanced pulmonary responses of peptide-specific CD4+/CD8+ Th1-like cells. These results provide first in vivo evidence implicating that early expansion/differentiation of Vγ2Vδ2 T effector cells during Mtb infection increases resistance to tuberculosis. Thus, data support a rationale for conducting further studies of the γδ T-cell-targeted treatment of established TB, which might ultimately help explore single or adjunctive phosphoantigen expansion of Vγ2Vδ2 T-cell subset as intervention of MDR-tuberculosis or HIV-related tuberculosis.

During the last several years, research has produced a significant amount of knowledge concerning the characteristics of human cd T lymphocytes. Findings regarding the immune functions of these cells, particularly their natural killer cell-like lytic activity against tumor cells, have raised expectations for the therapeutic applications of these cells for cancer. Pharmaceutical companies have produced selective agonists for these lymphocytes, and several teams have launched clinical trials of cd T cell-based cancer therapies. The findings from these studies include hematological malignancies (follicular lymphoma, multiple myeloma, acute and chronic myeloid leukemia), as well as solid tumors (renal cell, breast and prostate carcinomas), consisting of samples from more than 250 patients from Europe, Japan and the United States. The results of these pioneering studies are now available, and this short review summarizes the lessons learned and the role of cd T cell-based strategies in the current landscape of cancer immunotherapies.

In human blood, 1% to 5% of lymphocytes are ␥␦ T cells; they mostly express the ␥␦ T-cell receptor ( IntroductionThe success of therapeutic monoclonal antibodies (mAbs) in the treatment of cancer can be attributed to their multiple bioactivities. Their mechanism of action combines antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity, antibodydependent phagocytosis, direct cytotoxic activity, and inhibition of receptor signaling. 1 ADCC occurs when cytolytic effector cells expressing a receptor for the Fc region of the IgG class of antibodies (Fc␥ receptors) bind to antibodies on the surface of target cells. In humans, Fc␥ receptors comprise CD16 (Fc␥RIIIA-B), CD32 (Fc␥RIIA-C), and CD64 (Fc␥RI), which all bind the same region on IgG Fc but with low-to-medium (CD16, CD32) or high (CD64) affinities. 2 Several lines of evidence suggest that enhancing ADCC induced by therapeutic mAbs may directly improve their clinical efficacy. First, in mice bearing xenografted tumors, the efficacy of the therapeutic mAbs rituximab (RTX) and trastuzumab (TTZ) relies upon cell-surface expression of Fc␥R. 3 Second, ADCC is essential for the clinical efficacy of RTX in B-cell lymphoma patients and depends on the affinity of Fc␥RIIIA for the IgG. 4,5 Third, optimizing the affinity of RTX, TTZ, and alemtuzumab (ALZ) for Fc␥RIIIA increases their ADCC and their efficacy in preclinical and clinical studies. [6][7][8] Finally, recruitment and activation of additional cell effectors for ADCC might also enhance the cytolytic activity of anticancer mAbs. 9,10 The cytolytic effector cells involved in ADCC are CD16 ϩ (ie, Fc␥RIIIA)-positive natural killer (NK) cells and other CD8 ϩ cytolytic T lymphocytes, which release perforin through immunologic synapses to kill target cells. In addition, human CD4 Ϫ CD8 Ϫ ␥␦ T cells from peripheral blood might provide an important reservoir of cytolytic effector cells for ADCC. In most humans and nonhuman primate species, the majority of circulating ␥␦ T lymphocytes expresses the V␥9 T-cell receptor, with CD4 Ϫ CD8 Ϫ TCRV␥9 ϩ cells representing 1% to 3% of mononuclear cells. All these cells respond to stimulation with nonpeptide phosphoantigens (PAgs), which are small, phosphorylated metabolites produced by the cholesterol pathway in microbial pathogens and tumor cells. In addition to natural PAgs, the synthetic analog BrHPP 11 selectively stimulates TCRV␥9 ϩ ␥␦ T lymphocytes. PAg-stimulated ␥␦ T cells proliferate, secrete pro-inflammatory cytokines and chemokines, and, most importantly, kill leukemia, lymphoma, and carcinoma cells. 12,13 Several studies involving macaque monkeys 14,15 and clinical studies in cancer patients [16][17][18][19][20][21][22] have demonstrated in vivo the potential of PAg-activated TCRV␥9 ϩ ␥␦ T lymphocytes for cancer immunotherapy.The mechanism by which PAgs stimulate ␥␦ T cell-mediated cancer cell killing is unclear. The number of circulating ␥␦ T lymphocytes increases 50-to 100-fold in humans treated with BrHPP and IL2 (our unpublished observations...

IntroductionHuman ␥␦ T cells appear to belong to nonclassical T cells that contribute to both innate and adaptive immune responses. Circulating V␥2V␦2 (also termed V␥9V␦2) T cells exist only in primates and, in humans, constitute 60% to 95% of total blood ␥␦ T cells. V␥2V␦2 T cells in primates can be activated by nonpeptidic phosphorylated metabolites of isoprenoid biosynthesis (eg, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate [HMBPP], isopentenyl pyrophosphate [IPP], and its isomer dimethylallyl pyrophosphate [DMAPP]). [1][2][3] We have recently shown that HMBPP is associated with antigen-presenting cell (APC) membrane and specifically recognized by V␥2V␦2 T-cell receptor (TCR) expressed on V␥2V␦2 T cells. 4 Although HMBPP produced by mycobacteria/other microbes is a potent activator for V␥2V␦2 T cells, these ␥␦ T cells possess both innate and adaptive immune features. [5][6][7][8] The finding that "unprimed" V␥2V␦2 T cells can recognize and react to wide ranges of nonpeptide phospholigands with the capability of "naive" production of cytokines has been interpreted as a pattern recognition-like feature of innate immune cells. On the other hand, the capacity of V␥2V␦2 T cells to undergo major clonal expansion in primary infection and to mount rapid recall-like expansion upon reinfection has been proposed as adaptive immune response of these ␥␦ T cells. 8 Consistent with these memory-type responses is the demonstration of memory phenotypes of V␥2V␦2 T cells in the blood of humans. 9 Accumulating evidence suggests that V␥2V␦2 T cells play a role in mediating immunity against microbial pathogens 8 and tumors. 10 Foxp3-expressiong CD4 ϩ CD25 ϩ regulatory T cells (Tregs) control immune responses to self-antigens and foreign antigens and play a major role in maintaining the balance between immunity and tolerance. 11-14 Murine CD4 ϩ CD25 ϩ regulatory T cells are induced by transforming growth factor ␤ (TGF-␤), although TGF-␤ plus IL-6 favors the development of Th17 cells. 15 [23][24][25][26] We and others have also shown that IL-2 plus phospholigand treatment can induce remarkable expansion of V␥2V␦2 T cells in nonhuman primates. 1,27,28 We therefore took advantage of the IL-2-based in vivo model systems to assess potential interplay or mutual regulation between V␥2V␦2 T cells and Tregs during early mycobacterial infection in nonhuman primates. We found that phosphoantigenactivated V␥2V␦2 T cells were able to down-regulate IL-2-induced expansion of Tregs, and antagonize Treg-driven suppression of in vivo immune responses. Methods AnimalsFour-to 8-year-old, 3-to 4-kg cynomolgus macaques (Macaca fascicularis) were used in this study. A total of 18 monkeys were divided into 3 groups, 6 for each. All animals were maintained and used in accordance with the guidelines of the institutional animal care and use committee of all participating institutions. Animals were anesthetized with 10 mg/kg ketamine HCl (Fort Dodge Animal Health, Fort Dodge, IA) intramuscularly for all blood sampling and treatments. EDTA-anticoagulated...

Whereas αβ T cell receptors (TCR) recognize processed antigenic peptides or glycolipids bound respectively to major histocompatibility complex or CD1 molecules, γδ TCR react differently to a broad set of native antigens. A major human γδ T cell subset is activated through a mechanism involving Vγ 9Vδ 2 TCR and structurally unrelated phosphorylated nonpeptide antigens (referred to as phosphoantigens). Here, the structure‐function relationship of the strongest natural and synthetic phosphoantigens stimulating γδ cells was analyzed to elucidate the molecular basis of this unconventional recognition. Besides conformational determinants, we found that chemical reactivity of antigens is critical to their bioactivity. For Vγ 9Vδ 2 T cell activation, both organic and phosphorylated moieties of strong ligands undergo rapid and degradative structural changes. Conversely, analogs that are resistant to degradation specifically antagonize phosphoantigen‐mediated γδ T cell activation. These data suggest a novel mode of antigen perception involving both topological recognition and ligand consumption, which confers highly specific γδ T cell activation by structurally diverse ligands.

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Gammadelta-T-lymphocytes contribute to innate immunity and are selectively activated by nonpeptide phosphorylated molecules (so-called phosphoantigens) produced by organisms responsible for causing a broad range of infectious diseases. gammadelta-T-cells are also activated by synthetic phosphoantigens and are cytotoxic to tumor cells. Here we report the synthesis, NMR characterization, and comparative biological evaluation of new pyrophosphate, phosphonate, and pyrophosphonate monoesters whose structures correspond to isosteric analogues and stereoisomers of the highly potent isoprenoid metabolite ( E)-1-hydroxy-2-methylbut-2-enyl 4-diphosphate called HDMAPP (hydroxy-dimethyl-allyl pyrophosphate). Both pyrophosphate and pyrophosphonate series elicit promising gammadelta-T-cell stimulatory responses in vitro, the pyrophosphonate ester (C-HDMAPP) being by far more stable than its parent pyrophosphate ester (HDMAPP) with improved ADMET properties and a similar pharmacodynamic profile based on in vivo studies in nonhuman primate. In both series, we found that E-stereoisomers are the most active derivatives and that Z-stereoisomers show very marginal bioactivity levels. These results indicate that the use of bioisosteric analogues of HDMAPP may represent promising new leads for immunotherapy.