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Original article

Vγ9Vδ2 T lymphocytes activation in osteoporotic patients treated with bisphosphonates

Delia Sprini1 Laura Di Stefano1 Giovam Battista Rini1 Luisella Cianferotti2 Nicola Napoli 3

1

Department of Internal Medicine, University of Palermo, Italy 2 Department of Surgery and Translational Medicine, University of Florence, Florence, Italy 3 Division of Bone and Mineral Diseases, Washington University in St Louis, St Louis MO, USA

Address for correspondence: Giovam Battista Rini, MD, PhD Department of Medicine University of Palermo Palermo, Italy Phone: 0039 091 6552960 E-mail: [email protected]

Summary Treatment with bisphosphonates induces differentiation and activation of Vγ9Vδ2 T lymphocytes obtained from peripheral blood showing also an anti-tumoral effect in both in vitro and in vivo models. Aim of the present study was to determine in vivo the effect of BPs treatment in patients affected with osteoporosis on Vγ9Vδ2 T lymphocytes. We have studied Vγ9Vδ2 T lymphocytes expansion and differentiation from PBMC obtained from osteoporotic patients treated with one of the following bisphosphonates zoledronate, alendronate, neridronate or risedronate. We have found that zoledronic acid, followed by alendronate was the most effective on reducing CM population (100%) and increasing TEM and TEMRA γδ population. Our results indicate that in vivo treatment with BPs induces Vγ9Vδ2 cells to mature toward the effector phenotype, which may induce more antiresorptive responses. KEY WORDS: Vγ9Vδ2 T lymphocytes; osteoporosis; bisphosphonates.

Introduction Bisphosphonates (BPs) are the most used medications for the treatment of osteoporosis and bone metastases (1). BPs are synthetic stable analogues of inorganic pyrophosphate (Ppi),

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have high affinity for calcium in bone mineral matrix, where they are internalized by osteoclasts causing their inactivation. BPs have a wide range of target cells including macrophages, osteoblasts, enterocytes, tumor and stromal cells (1, 2). BPs recognize as targets the enzymes of the mevalonate pathway, required for the synthesis of cholesterol and isoprenoid lipids (3). These isoprenoid residues are necessary for prenylation of the small GTPases, important signaling proteins. Several studies have shown that the BPs also have antitumor potentials, an effect played not only in bone metastases but also in multiple myeloma (4, 5). These compounds have the ability to activate Vγ9Vδ2 T lymphocytes and their proliferation produces prophlogistic cytokines like Interferon gamma (IFN) and Tumor Necrosis Factor (TNF) alpha; in addition, this subset display efficient cytotoxic activity against neoplastic cells (6). This effect was observed in both in vitro (4), and in vivo models (7). Treatment with zoledronic acid of patients with prostate and breast cancer reduced effect of bone metastases and induced differentiation and activation Vγ9Vδ2 T lymphocytes obtained from peripheral blood (5). We have already shown in vitro that treatment with BPs induces Vγ9Vδ2 T lymphocytes to differentiate towards an effector/cytotoxic phenotype showing also a major content of IFN-gamma (8). Aim of the present study was to determine in vivo the effect of BPs treatment in patients affected with osteoporosis on Vγ9Vδ2 T lymphocytes.

Methods Study subjects Thirty-five Caucasian female patients were recruited and randomized to receive alendronate (ALE), zoledronate (ZOL), risedronate (RIS), neridronate (NER). Those on ALE (11) and RIS (11) were treated for three months with a weekly oral dose of 70 mg and 35 mg respectively. Those on NER (6) underwent an IM administration for 3 months while those on ZOL had one 5 mg IV infusion. At the baseline, patients received a physical examination, DEXA measurement (L1-L4 and proximal femur), and blood draw. 3 months after the beginning of treatment patients were recalled for blood draw and serum was collected. Biochemistry Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation of heparinezed peripheral blood over FicollHypaque gradients (Euroclone, UK). Vγ9Vδ2 T lymphocytes cell expansion and differentiation was evaluated after one week of culture. For subset identification, PBMC were isolated and analyzed using 3 color flow cytometry (FACScan, Germany). Mononuclear antibodies used included: fluorescein isothiocynate (FITC)-conjugated anti VD2, phycoerithrin (PE)Clinical Cases in Mineral and Bone Metabolism 2014; 11(2): 126-128

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Vγ9Vδ2 T lymphocytes activation in osteoporotic patients treated with bisphosphonates conjugated anti CD27 and PE Cy5-conjugated anti CD 27 and PE Cy5-conjugated anti-CD45RA. Viability was confirmed by trypan blue exclusion test. The absolute number of Vγ9Vδ2 T cells present in each colture was calculated according to the a formula previously published (8). After one week of colture, Vγ9Vδ2 T lymphocytes were isolated by positive selection using hapten-modified TCR γδ antibody and FITC-conjugated antihapten microbeads (MACS, Germany). Cells so obtained were analyzed by FACS analysis and were more than 98% Vγ9Vδ2 T lymphocytes CD3 as determined by FACS analysis.

Results Clinical features of study subjects are shown in Table 1. All patients were affected with osteoporosis and there were no differences in terms of age or T-score between the 4 groups. Analysis of the effect of the treatment on different subgroups of Vγ9Vδ2 T lymphocytes is shown in Table 2. Treatment with ZOL reduced by 85% the T NAÏVE subpopulation, followed by ALE, RIS and NER. ZOL showed also the highest reduction in terms of CM population (100%) while patients treated with RIS had more than 50% less reduction compared to ZOL. TEM and TEMRA γδ were increased by 100% in ZOL patients, followed by ALE, NER and RIS.

Discussion Vγ9Vδ2 T lymphocytes have important effects in the local immunity vs infectious pathogens but recent studies show also well recognized citotoxic activity against tumor cells (4, 5). BPs are the most used medications for treatment of osteoporosis and their antifracture efficacy has been widely proven (1, 2). Recent animal studies have shown that the BPs, have also potent antiangiogenic activity that may contribute to their anti-resorptive effect (9). Thus, there is increasing evidence that BPs, especially the nitrogen-containing compounds, can lead to direct and indirect effects that result not only in less bone loss but less tumor

burden as well (9). In the present study we have found that all tested BPs caused a substantial differentiation of Vγ9Vδ2 T lymphocytes toward an effector-memory-like phenotype. Osteoporotic patients treated with ZOL had the highest decrease of the absolute numbers of Naïve and CM cells while peripheral TEM and TEMRA γδ cells were strongly increased. TEMRA are part of the Vγ9Vδ2 pool and play a main role against tumor cells and intracellular bacteria. They represent the most differentiated type of Vγ9Vδ2 cells and have the most effective antitumoral effect. We here confirm our previous in vitro findings showing that the BPs, in particular Zol, treatment enhance Vγ9Vδ2 differentiation (8). All together, our results indicate that in vivo treatment with BPs induces Vγ9Vδ2 cells to mature toward an IFNγ-producing effector phenotype, which may induce more antiresorptive responses. Pharmacokinetics of these drugs differ among them and it may reflect also the differences in terms of Vγ9Vδ2 activation. In fact, ZOL is the most powerful BP, followed by ALE, while risedronate holds a low affinity to bone matrix. Here we describe a similar trend, showing that ZOL and ALE have the highest efficacy on causing the drop of Naïve and CM populations and enhancing the TEM and TEMRA γδ cells populations. These last ones act in the sites of inflammation where they display immediate effector functions such as cytokine production and citotoxicity (10). It should be considered that both ZOL and ALE have the most validated antifracture efficacy but, at the same time, have been investigated given possible side effects as osteonecrosis of the jaw (ONJ) and atypical femoral fractures (11). If their role on Vγ9Vδ2 T lymphocytes cells contribute to the onset of these complications is worth of investigation. At the same time this mechanism has been well studied in terms of tumor immunotherapy (12). These drugs were shown to induce expansion of Vγ9Vδ2 T cells in peripheral blood mononuclear cell cultures and enhance cytotoxicity of malignant plasma cells in bone marrow cultures. This mechanism of action is the base for new therapeutic approaches and current clinical trials (13, 14).

Table 1 - Clinical features of the study subjects. Age

t-score L1-L4

t-score fem. neck

Zoledronate (7)

64 ± 8

-2.5 ± 0.8

2.0 ± 0.8

Alendronate (11)

65 ± 10

-2.7 ± 0.8

-2.93 ± 0.8

Neridronate (9)

63 ± 5

-2.6 ± 0.8

-2.4 ± 0.6

Risedronate (11)

62 ± 9

-2.2 ± 0.5

-2.2 ± 0.7

Table 2 - Effect of different BPs on Vγ9Vδ2 T lymphocytes subpopulations. NAÏVE

CM

EM

TEMRA

Zoledronate

▼ 72%

▼63%

▲72%

▲90%

Alendronate

▼ 45%

▼45%

▲63%

▲72%

Neridronate

▼ 66%

▼66%

▲66%

▲66%

Risedronate

▼ 85%

▼100%

▲100%

▲100%

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D. Sprini et al. In conclusion, the activation of peripheral blood Vγ9Vδ2 T cells by BPs may explain part of their pleiotropic effects. Given the advances on the use of these medications in cancer treatment, our data may shed some light also on their effects in antiresorptive efficacy in osteoporotic patients.

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