## 2 Expansion Strategies
### \(\gamma\delta\) T Cell Modulation with Different Substances
#### 2.4.1 Interleukins  
IL-2 cytokine has been most frequently used for supporting the expansion and survival of V\(\gamma\)9V82 T cells [107]. Its working concentration ranged from 100 to 300 IU when combined with pAsg and from 100 to 1000 IU when used with ZOL; and it requires repetitive replenishment in the culture media every 2-3 days [46; 98]. Growing evidence indicates that IL-15 is more efficient at expanding \(\gamma\)8 T cells with an effector phenotype compared to IL-2. Moreover, effector memory V\(\gamma\)9V82 T cells derived from renal cell carcinoma tumors were efficiently expanded using BrHPP combined with IL-15, but not IL-2 [108]. In addition, IL-15 induced higher toxicity in BrHPP activated V\(\gamma\)9V82 T cells against different adherent tumor cells compared to IL-2. This was further supported by the IL-15-mediated overexpression of surface CD56 [109] which is believed to be a marker of \(\gamma\)8 T cells cytotoxicity [110]. Similarly, IL-15 in combination with IL-2, boosted the proliferation as well as the in vitro anti-tumor activity of ZOL-expanded V\(\gamma\)9V82 T cells [111]. Taken together, these studies encourage the use of IL-15 instead of, or in combination with, IL-2 for the expansion of V\(\gamma\)9V82 T cells, especially with a boosted effector potential, which may enhance the therapeutic efficacy of the V\(\gamma\)9V82 T cell. IL-21, a cytokine previously known to enhance NK and CD8\({}^{+}\) T cell cytotoxicity, was found to significantly promote the proliferation of pAg-induced V\(\gamma\)9V82 T cells in a dose-dependent manner. IL-21 was shown to enhance proinflammatory response and anti-tumor cytolytic activity of \(\gamma\)8 T cells [112]. It also promotes \(\gamma\)8 T cells mediated B cell maturation [48]. V\(\gamma\)9V82 T cells from AML patients exhibited lower expression of IL-21R, which affected their response to IL-21. This was confirmed by the need for a higher dose of IL-21 for expansion and lower increase in STAT1 phosphorylation compared to V\(\gamma\)9V82 T cells from healthy volunteers. Remarkably, AML V\(\gamma\)9V82 T cells displayed significantly higher Tim-3 expression compared to healthy V\(\gamma\)9V82 T cells which was further intensified by pAsg and IL-2. [113]. Interestingly, Tim-3 blockade could restore the proliferation and the STAT1 phosphorylation in V\(\gamma\)9V82 T cells in response to IL-21. These data indicate that IL-21  
Figure 3: \(\gamma\)8 T cells modulation using different substances. Multiple cytokines such as IL-2, IL-7, IL-15, IL-21 and vitamin C skew the profile of \(\gamma\)8 T cells toward Th1-like profile meaning increased proliferation, survival, and cytotoxicity. Transforming growth factor-\(\beta\) (TGF-\(\beta\)) enhances Th1-like profile, as well as inducing \(\gamma\)8 T cells migration and synapse formation with target cells. Monoclonal antibodies (mAb) and bispecific Abs targeting a tumor associated antigen when used in combination with \(\gamma\)8 T cell therapy, can direct \(\gamma\)8 T cells to the tumor cells and enhance the formation of cell-cell immunological synapse leading to increased cytotoxicity. Ab, antibody; Vit. C, vitamin C.  
could significantly expand the V\(\gamma\)9V\(\delta\)2 T cells, but its potency was restricted due to simultaneously increasing the expression of checkpoint inhibitor, Tim-3 [57]. The central role of interleukin-7 (IL-7) in maintaining the T cell homeostasis is well established [114]. IL-7 exert a pivotal role in modulating \(\gamma\)8 T cells function by (a) controlling homeostasis and tissue distribution in both human and mice [115], (b) supporting the development and survival of cutaneous resident \(\gamma\)8 T cell population [116] and (c) upregulating \(\gamma\)8 T cell's BTLA expression with consequent apoptosis induction [117]. Moreover, IL-7 is associated with selective expansion of IL-17-producing \(\gamma\)\(\delta\) T cells [118].