Highly Active Microbial Phosphoantigen Induces Rapid yet Sustained MEK/Erk- and PI-3K/Akt-Mediated Signal Transduction in Anti-Tumor Human γδ 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

Peripheral blood T-cell signatures from high-resolution immune phenotyping of γδ and αβ T-cells in younger and older subjects in the Berlin Aging Study II

Background Aging and latent infection with Cytomegalovirus (CMV) are thought to be major factors driving the immune system towards immunosenescence, primarily characterized by reduced amounts of naïve T-cells and increased memory T-cells, potentially associated with higher morbidity and mortality. The composition of both major compartments, γδ as well as αβ T-cells, is altered by age and CMV, but detailed knowledge of changes to the γδ subset is currently limited. Results Here, we have surveyed a population of 73 younger (23–35 years) and 144 older (62–85 years) individuals drawn from the Berlin Aging Study II, investigating the distribution of detailed differentiation phenotypes of both γδ and αβ T-cells. Correlation of frequencies and absolute counts of the identified phenotypes with age and the presence of CMV revealed a lower abundance of Vδ2-positive and a higher amount of Vδ1-positive cells. We found higher frequencies of late-differentiated and lower frequencies of early-differentiated cells in the Vδ1+ and Vδ1-Vδ2-, but not in the Vδ2+ populations in elderly CMV-seropositive individuals confirming the association of these Vδ2-negative cells with CMV-immunosurveillance. We identified the highest Vδ1:Vδ2 ratios in the CMV-seropositive elderly. The observed increased CD4:CD8 ratios in the elderly were significantly lower in CMV-seropositive individuals, who also possessed a lower naïve and a larger late-differentiated compartment of CD8+ αβ T-cells, reflecting the consensus in the literature. Conclusions Our findings illustrate in detail the strong influence of CMV on the abundance and differentiation pattern of γδ T-cells as well as αβ T-cells in older and younger people. Mechanisms responsible for the phenotypic alterations in the γδ T-cell compartment, associated both with the presence of CMV and with age require further clarification

Zoledronic acid renders human M1 and M2 macrophages susceptible to Vδ2(+) γδ T cell cytotoxicity in a perforin-dependent manner.

Vδ2(+) T cells are a subpopulation of γδ T cells in humans that are cytotoxic towards cells which accumulate isopentenyl pyrophosphate. The nitrogen-containing bisphosphonate, zoledronic acid (ZA), can induce tumour cell lines to accumulate isopentenyl pyrophosphate, thus rendering them more susceptible to Vδ2(+) T cell cytotoxicity. However, little is known about whether ZA renders other, non-malignant cell types susceptible. In this study we focussed on macrophages (Mϕs), as these cells have been shown to take up ZA. We differentiated peripheral blood monocytes from healthy donors into Mϕs and then treated them with IFN-γ or IL-4 to generate M1 and M2 Mϕs, respectively. We characterised these Mϕs based on their phenotype and cytokine production and then tested whether ZA rendered them susceptible to Vδ2(+) T cell cytotoxicity. Consistent with the literature, IFN-γ-treated Mϕs expressed higher levels of the M1 markers CD64 and IL-12p70, whereas IL-4-treated Mϕs expressed higher levels of the M2 markers CD206 and chemokine (C-C motif) ligand 18. When treated with ZA, both M1 and M2 Mϕs became susceptible to Vδ2(+) T cell cytotoxicity. Vδ2(+) T cells expressed perforin and degranulated in response to ZA-treated Mϕs as shown by mobilisation of CD107a and CD107b to the cell surface. Furthermore, cytotoxicity towards ZA-treated Mϕs was sensitive-at least in part-to the perforin inhibitor concanamycin A. These findings suggest that ZA can render M1 and M2 Mϕs susceptible to Vδ2(+) T cell cytotoxicity in a perforin-dependent manner, which has important implications regarding the use of ZA in cancer immunotherapy

Effect of Longâ Term Oral Bisphosphonates on Implant Wound Healing: Literature Review and a Case Report

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141603/1/jper0584.pd

Bu-Shen-Ning-Xin decoction: inhibition of osteoclastogenesis by abrogation of the RANKL-induced NFATc1 and NF-κB signaling pathways via selective estrogen receptor α

Ling Wang,1,2,* Xue-Min Qiu,1,2,* Yu-Yan Gui,1,2 Ying-Ping Xu,1,2 Hans-Jürgen Gober,3 Da-Jin Li11Laboratory for Reproductive Immunology, Hospital and Institute of Obstetrics and Gynecology, IBS, Fudan University Shanghai Medical College, 2Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Shanghai, People’s Republic of China; 3Department of Pharmacy, Wagner Jauregg Hospital and Children’s Hospital, Wagner Jauregg Weg, Linz, Austria*These authors contributed equally to this workIntroduction: Bu-Shen-Ning-Xin decoction (BSNXD) is a traditional Chinese medicinal composition that has been used as a remedy for postmenopausal osteoporosis, but the mechanisms affecting bone metabolism are not fully understood.Purpose: We investigated the molecular mechanism and signaling pathway underlying the effect of BSNXD on osteoclastogenesis.Materials and methods: A postmenopausal osteoporosis animal model generated by ovariectomy was administered BSNXD and drug-derived serum was prepared. An enzyme immunoassay was conducted to measure the 17-β-estradiol (E2) concentration in the drug-derived serum. Bone marrow-derived monocyte/macrophage precursor cells were treated with drug-derived serum, and tartrate-resistance acid phosphatase staining was conducted to observe osteoclastogenesis. A bone resorption assay was performed to analyze the effect on osteoclastic resorptive function. Real-time PCR, flow cytometry, Western blotting, transfection, and luciferase assays were conducted to explore the related mechanism.Results: E2 was not elevated in BSNXD-derived serum. BSNXD-derived serum suppressed receptor activation of nuclear factor κB ligand (RANKL)-activated osteoclastogenesis in a dose-dependent manner; this effect could be reversed by estrogen receptor α antagonist methyl-piperidino-pyrazole. The serum suppressed RANKL-induced NF-κB transcription and inhibited the accumulation of nuclear factor of activated T-cells, cytoplasmic 1 in osteoclast precursor cells; the inhibitory effect was abolished by methyl-piperidino-pyrazole but not the estrogen receptor β antagonist or androgen receptor antagonist.Conclusion: These results collectively suggest that administration of BSNXD presents inhibitory effects on osteoclast differentiation by abrogating the RANKL-induced nuclear factor of activated T-cells, cytoplasmic 1 and NF-κB signaling pathways downstream of estrogen receptor α, thereby contributing to the inhibitory effect on bone resorption.Keywords: herbal formula, osteoclastogenesis, estrogen receptor α, NF-κB, NFATc

Bu-Shen-Ning-Xin Decoction ameliorated the osteoporotic phenotype of ovariectomized mice without affecting the serum estrogen concentration or uterus

Ling Wang,1,2,* Xue-Min Qiu,1,2,* Yu-Yan Gui,1,2 Ying-Ping Xu,1,2 Hans-Jürgen Gober,3 Da-Jin Li1 1Laboratory for Reproductive Immunology, Hospital and Institute of Obstetrics and Gynecology, IBS, Fudan University Shanghai Medical College, 2Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Shanghai, People’s Republic of China; 3Department of Pharmacy, Wagner Jauregg Hospital and Children’s Hospital, Linz, Austria *These authors contributed equally to this work Introduction: Bu-Shen-Ning-Xin Decoction (BSNXD), a traditional Chinese medicinal composition, has been used as a remedy for postmenopausal osteoporosis, but its effects on bone metabolism and the uterus have not been reported.Purpose: We aimed to determine the respective effects of BSNXD on the bones and the uterus of ovariectomized (OVX) mice to evaluate the efficacy and safety of this herbal formula.Materials and methods: Postmenopausal osteoporosis animal models that were generated by ovariectomy were treated with BSNXD. Dual-energy X-ray absorptiometry was performed to analyze the bone mineral density, and histomorphometric analysis was performed to measure the parameters related to bone metabolism. Calcein labeling was performed to detect bone formation. The uteruses from the mice were weighed, and the histomorphometry was analyzed. Drug-derived serum was prepared to assess the 17-β-estradiol concentration via enzyme immunoassay.Results: BSNXD administration ameliorated the osteoporotic phenotype of OVX mice, as evidenced by an increase in the bone mineral density and bone volume; these effects could not be abolished by the administration of the aromatase inhibitor letrozole. Moreover, BSNXD had no effect on the serum estrogen concentration or uterus.Conclusion: These results suggest that BSNXD has ameliorating effects on bone loss due to estrogen deprivation without affecting the peripheral blood estrogen concentration or the uterus in OVX mice. Keywords: traditional Chinese medicine, postmenopausal osteoporosis, OVX, bone phenotype, estrogen&nbsp