## 2 Expansion Strategies
### Ex Vivo Expansion of V\(\delta\)2 \(\gamma\delta\) T Cells  
Using pAg is the most established method for expanding V\(\delta\)2 \(\gamma\delta\) T cells in vitro and in vivo [17]. The most potent pAg activators for V\(\delta\)2 T cells are isopentenyl pyrophosphate (IPP) and (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), the latter being a natural intermediate of the non-mevalonate pathway of IPP biosynthesis [78; 79; 80]. Small changes in the structure of these pAg, either on the phosphate or the isoprene moieties, profoundly affect the \(\gamma\delta\) T cell stimulatory capacity. Several analogs of these compounds have been synthesized with an intermediate stimulatory capacity between that of IPP and of HMBPP [81]. The recognition of tumors by \(\gamma\delta\) T cells is ascribed to the abnormally elevated production of IPP by tumor cells, as result of changes in the regulation of their isoprenoid metabolic pathway [82]. These findings support the concept that \(\gamma\delta\) T cells may cross-react to phosphorylated metabolites accumulating inside tumor cells and to metabolites released by bacterial cells in the microenvironment. Importantly, when bacteria infect target cells, they induce alteration of the host isoprenoid pathway by subverting several regulatory mechanisms [83; 84]. These alterations lead to a transient and acute accumulation of IPP, which is then responsible for the activation of \(\gamma\delta\) T cells. Therefore, summarizing, \(\gamma\delta\) T cells may recognize (i) tumor cells that accumulate IPP; (ii) bacterial metabolites such as HMBPP, and (iii) cells accumulating IPP following infection with bacteria not producing HMBPP (Figure 2). Figure 2: Various phosphoantigen and bisphosphonate compounds. Some of the most important pAgs and BPs mentioned in the text are shown here. BPs are a class of chemical compounds with two PO\({}_{3}\) (phosphonate) groups that are widely used to treat osteoporosis (the condition of low bone density). Zoledronate is one of the most potent N-BPs that have been widely used for in vitro and in vivo expansion of V\(\delta\)2 \(\gamma\delta\) T cells. (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) is an essential metabolite in most pathogens including mycobacterium tuberculosis and malaria. Isopentenyl pyrophosphate (IPP) and geranyl pyrophosphate (GPP) are the intermediate metabolites in the isoprenoid biosynthesis pathways. Bromohydrin pyrophosphate (BrHPP) is a synthetic alkyl1 diphosphate pAg. Pyrophosphates such as IPP and GPP are able to directly stimulate V\(\delta\)2 \(\gamma\delta\) T cells, while BPs act indirectly via blocking the FPPS enzyme in isoprenoid biosynthesis pathways which results in IPP and GPP accumulation in the cells (see Figure 1). BPs were originally used in the treatment of osteoporosis and other bone disorders [85]; however, they have shown to prompt several clinical indications including cytotoxic activity as a monotherapy [64] and activation of \(\gamma\delta\) T cells [86]. Zoledronic acid (Zoledronate, Zometa, ZOL) is the most potent of the clinically used N-BP [87]. ZOL has direct effects on tumor cells in vitro through induction of apoptosis and reduction of viability and proliferation of several cancer cell lines [88]. The various natural and synthetic pAg compounds are shown in Figure 2.  
Though the molecular recognition events are not yet fully explained, it seems that a hydrophobic alkyl moiety linked to a polar group containing one or more phosphates in these small chemical compounds, is sensed by \(\gamma\delta\) T cells consequently activating \(\gamma\delta\) T cells in a TCR-dependent manner [89]. It has been reported that BPs block the isoprenoid biosynthesis pathway by inhibiting famesyl pyrophosphate synthase (FPPS) enzyme and cause the accumulation of prenyl pyrophosphate metabolites, such as IPP and geranyl pyrophosphate (GPP) within cells [90]. IPP and GPP bind to the cytoplasmic tail of a B7-family related molecule of the Ig-superfamily, called butylrophilin 3A1 (BTN3A1) [91].