## 2 Expansion Strategies  
The broad application of \(\gamma\delta\) T cells for adoptive cell immunotherapy has been hindered by their low physiological frequency in the periphery, and the difficulty of ex vivo expansion. Considerable efforts are currently devoted to developing suitable methods for obtaining clinical numbers of \(\gamma\delta\) T cells [45]. The expansion strategy of \(\gamma\delta\) T cells can be bimodal: ex vivo and in vivo. In the first, \(\gamma\delta\) T lymphocytes are isolated from peripheral blood mononuclear cells (PBMCs) and stimulated ex vivo using synthetic phosphoantigen (pAg) or bisphosphonates (BP) such as zoledronic acid [46]. Ex vivo expansion of \(\gamma\delta\) T cells has been clinically applied and has shown promising results [41]. The second approach involves stimulation and expansion of \(\gamma\delta\) T cells in vivo by systemic administration of pAg or nitrogenous-BP (N-BP). These two approaches will be explained in detail in the following section (Tables 1 and 2). \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**pAg or BP (conc.)** & **Additional Stimuli** & **Cytekine (conc.)** & **Transduction** & **Subset** & **Target** & **Citation** \\ \hline ZOL US (sub) & & IL-2 (100 IU/mL) & - & - & - & [PERSON]. 2020 [47] \\ Synthetic HMBP (0.1-1.0 & IL-2, IL-4, IL-7, IL-15, & - & V\({}_{9}\)V9X2 & - & [PERSON] 2007 [48] \\ nM) & & IL-21, IFN-8 etc. & - & V\({}_{9}\)V9X2 & - & [PERSON]. 2007 [48] \\ IPP (2 ug/mL) & & IL-21/LL-4 & - & - & - & [PERSON]. 2001 [49] \\ ZOL (5 uM) & & IL-20 (200 IU/mL) & - & - & Cholanglocari-noma & [PERSON]. 2018 [50] \\ HMBP (0.1-10 mg/mL) & & IL-2, IL-7, IL-15, IL-21 & - & - & [PERSON]. 2002 [51] \\ IPP (variable) & & IL-2, IL-7, IL-15 & - & V\({}_{9}\)V9X2 & - & [PERSON] [PERSON]. 2005 [52] \\ IPP (50 uM) & & aAPC, anti-y\(\times\)T & IL-2, IL-21 & - & Polyclonal & Neuroblastoma & [PERSON]. 2014 [53] \\ Pamidronate (10 ug/mL) & & IL-2, IL-23, IL-16, IL-6 & - & - & - & [PERSON]. 2020 [54] \\ ZOL (5 uM) & & IL-21, IFN-8 etc. & - & V82, V81 & - & [PERSON]. 2019 [55] \\ PM/Momomomom (750 & & - & - & V82, V81 & - & [PERSON]. 2019 [55] \\ ng/mL) & & Anti-y\(\times\) TCR mAb & IL-2, IL-15 & - & V82 & - & [PERSON] [PERSON]. 2020 [56] \\ IZOL, IP(20 mg/mL) & & Feeder cells & IL-2, IL-21 & Retroviral & V9V82 & - & [PERSON]. 2019 [57] \\ IPP (2-5 ug/mL) & & IL-2 (100-1000 U/mL) & Lattiviral & - & - & [PERSON]. 2019 [58] \\ ZOL (5 uM) & & IL-20 (100-2000 U/mL) & Retroviral & - & - & [PERSON]. 2019 [59] \\ ZOL (40 ug/mL), Con-A & & IL-2, IL-4 & Retroviral & V82, V81 & - & [PERSON]. 2017 [42] \\ ZOL (5 uM), OKT3 & & IL-2(1000 IU/mL) & RNA electroporation & Melanoma & Harrer DC. 2017 [60] \\ ZOL (1 ug/mL) & & IL-2 (100 IU/mL), & Retroviral & - & - & [PERSON]. 2004 [61] \\ ZOL (1 uM) & & IL-2 (200 IU/mL) & Lattiviral & - & Glioblastoma & Lamb IS. 2013 [62] \\ ZOL (5 uM) & & IL-2 (200 IU/mL) & RNA electroporation & V\({}_{9}\)V82 & - & [PERSON]. 2015 [63] \\ ZOL (5 uM) & & Engineerd K526 & IL-2 (200 IU/mL) & RNA electroporation & V\({}_{9}\)V82 & - & [PERSON]. 2018 [45] \\ ZOL, 5 uM) & & IL-2 (200 IU/mL) & RNA electroporation & V\({}_{9}\)V82 & - & [PERSON]. 2018 [45] \\ \hline ZOL,,solderonate; HMBP7; (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate; IPP, Isopenrnyi pyrophosphate; Con-A, concanavalim-A; mAb, monoclonal antibody. \({}^{*}\) Used umbilical \\ card as source. & & & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: In vitro studies using phosphoantigens or bisphosphonates for \(\gamma\)$ T cells expansion. \begin{table}
\begin{tabular}{c|c c c c c} \hline
**PAS or BP (conc. )** & **Treatment Strategy** & **Cytokine** & **Subset** & **Target** & **Citation** \\ \hline ZOL (0.05 mg/kg.1-3 doses) & IV infusion, then in vitro & - & V\(\otimes\)2, V\(\otimes\)1 & Leukemia & Bertaina A. 2017 [37] \\ ZOL (4 mg starting dose) & \begin{tabular}{c} IV infusion + \\ chemotherapy \\ \end{tabular}  & - & - & Breast cancer & AfR.