## 2 Expansion Strategies
### In Vivo (Systemic) Expansion of \(\gamma\delta\) T Cells  
\(\gamma\delta\) T cells have complex biology and tissue specific tropism; therefore, they may not behave in in vitro culture as they would under physiologic conditions. This makes interpreting the results of \(\gamma\delta\) T cells from in vitro studies challenging [128]. To harness the \(\gamma\delta\) T cell anti-tumor activity, many researchers have attempted to activate \(\gamma\delta\) T cells in vivo for the treatment of a broad range of tumors. [PERSON] et al. have shown that systemic administration of ZOL, to expand V\(\delta\)2 \(\gamma\delta\) T cells in vivo, resulted in increased cytotoxic anti-tumor ability of V\(\delta\)2 cells and better overall outcomes in transplanted pediatric leukemia patients. From a clinical point of view, patients who received repeated infusion of ZOL (three or more) showed better survival when compared to those who received 1-2 ZOL infusions [37]. The authors analyzed the effects of ZOL on \(\gamma\delta\) T cells in vivo, using classical phenotypical and functional assays, synergistically integrated with innovative proteomic tools of sample preparation, as well as analytical conditions including high-resolution mass spectrometry, statistical and network analysis. These novel proteomic approaches applied to clinical studies and high-resolution mass spectrometry revealed an in vivo evolution of \(\gamma\delta\) T cell proteotype mediated by ZOL. In particular, proteomic analysis of \(\gamma\delta\) T cells purified from patients showed upregulation of proteins involved in activation processes and immune response, paralleled by downregulation of proteins involved in proliferation. Such effects were already evident after the first ZOL infusion but were further boosted by the subsequent infusions. These outcomes mirrored the phenotypic changes observed through flow cytometry in both V\(\delta\)1 and V\(\delta\)2 subsets. ZOL influenced, unexpectedly, the phenotype and function not only of V\(\delta\)2 cells, which selectively recognize pAgs, but also of the V\(\delta\)1 population [37].  
Administration of pAgs or N-BP in combination with IL-2 has been a common strategy for expanding V\(\delta\)2 \(\gamma\delta\) T cells in the body. Systemic administration of BrHPP or N-BP (ZOL or pamidronate), in combination with IL-2 has been tested in more than eight clinical trials so far and results have proven safety of the treatment. A successful expansion and maturation of V\(\delta\)2 T cells towards IFN-\(\gamma\)-producing effector phenotype was observed in most patients [41].  
Data has also demonstrated that IL-2 plays a critical role in the in vivo expansion of \(\gamma\delta\) T cells. In a study by [PERSON] et al. ZOL alone or in combination with low-dose IL-2 was used for in vivo expansion of \(\gamma\delta\) T cells in metastatic hormone-refractory prostate cancer. The results showed no expansion of \(\gamma\delta\) T cells in the group treated with ZOL alone, whereas in the group treated with ZOL plus IL-2, 5/9 patients showed an increase in \(\gamma\delta\) T cells population and improved clinical outcome [66]. Some of the clinical studies focused on activating \(\gamma\delta\) T cells via systemic infusion of pAgs and BPs or adaptive transfer of pAg- and BP-expanded \(\gamma\delta\) T cells for cancer therapy are listed in Table 2.