Expansion strategies for Vδ2 γδT cells in cancer immunotherapy: Activation, cytokines, and culture conditions

Table 1 Summary of the cytokine milieu used in in-vitro expansion of human γδT cells stimulated with N-aminobisphosphonates or phosphoantigens

Cytokine	Impact of cytokine on Vδ2 T expansion		Concentration	Ref.
Common gamma chain
IL-2	+	Supports robust γδT cell proliferation and enhances cell viability during extended in vitro culture. Drives the cytotoxic phenotype, induces CD107a, IFN-γ, and TNF-α expression in a dose-dependent manner, indicating enhanced degranulation and effector function. Upregulation of early activation markers, such as CD69 and CD25, as well as molecules involved in adhesion and cytotoxicity (LFA-1, ICAM-1, NKG2D, CD94). It promotes a proinflammatory effector phenotype (CD27-, CD28-) and CXCL10, but also an increased expression of KLRG1, 2B4. Upregulate expression of Th1-type cytokines and effector molecules, including IFN-γ, TNF-α, GM-CSF, CCL3, IL-5, and IL-13	100-1000 IU/mL	Kondo et al[5], Nada et al[21], Peters et al[51], Mariani et al[52], Li and David Pauza[53]
IL-4	+	Enhances the proliferation, however, weaker than IL-2 and IL-15. Upregulation of early activation markers, e.g. CD69, and LFA-1, ICAM-1, NKG2D, CD94, CD27, CD269	10 ng/mL	Vermijlen et al[26]
IL-9	+/-	Enhances the expression of IL-9R mRNA. Upregulates IL-17 and IFN-γ expression	2 ng/mL	Guggino et al[11]
IL-15	+	Promotes γδT cell proliferation and viability, even at low concentrations. Improves survival by increasing Bcl-2 expression and downregulating apoptotic mediators such as caspases 3 and 8, thereby supporting long-term maintenance of γδT cells in culture. Enhances cytotoxic potential by promoting the production and secretion of granzyme B, perforin, and granulysin. Increases the proportion of CD56+ and CD16+ γδT cells. Induces early activation markers, including CD25 and CD69. Favours the development of an early/central memory phenotype (CD27+, CD28+/-) and, when combined with IL-2, further enhances the expression of CCR9, HLA-DR, CD86, and CD70. Notably, while IL-15 does not significantly modulate the surface levels of CD16, CD56, NKG2D, DNAM-1, PD-1, or other natural cytotoxicity receptors, it functionally boosts downstream effector pathways. IL-15-stimulated γδT cells exhibit robust IFN-γ secretion, reportedly higher than in traditional IL-2-based protocols, while lacking expression and secretion of IL-10, IL-13, and IL-17, suggesting a Th1-skewed, non-regulatory profile	2-40 IU/mL	Aehnlich et al[18], Dunne et al[19], Sawaisorn et al[20], Nada et al[21], Burnham et al[22], Vermijlen et al[26], Peters et al[43], Chen and Freedman[54], Tyler et al[55], Izumi et al[56], Guo et al[57]
IL-21	+/-	Does not support long-term proliferation of γδT cells following phosphoantigen activation. However, it can enhance expansion in “non-expanders”, i.e., donors who respond poorly to conventional protocols. Enhances cytotoxic potential by upregulating CD107a expression and promoting degranulation. Increases expression of key effector molecules, including granzyme A, granzyme B, and perforin. IL-21 induces early activation markers (CD25, CD69) and upregulates surface molecules involved in adhesion and cytotoxicity, such as CD56, LFA-1, ICAM-1, NKG2D, CD94, and CXCL10. Favours an effector memory phenotype and upregulates regulatory and exhaustion-associated molecules (CD244, CD152, KLRG1). Selectively induces expression of CD73 and overexpression of CXCL13. Promotes a proinflammatory Th1-skewed response, though weaker than IL-2. It stimulates secretion of IFN-γ almost exclusively, but also induces IL-8 and IL-10 production.	10-30 ng/mL	Burnham et al[22], Barjon et al[23], Thedrez et al[24], Vermijlen et al[26]
Others
IL-7	+/-	Enhances expanded γδT cells viability. No effect on the induction of naive CD4+ Vγ9/Vδ2 T cells proliferation. Does not induce HLA-DR expression.	20-50 ng/mL	Tyler et al[55]
IL-12	+/-	The effects of IL-12 on proliferation are inconsistent. Some studies report limited impact on cell expansion when IL-12 is used alone. However, in specific contexts, particularly when TNF-α is present, IL-12 may support expansion without the need for IL-2. May have dual effects on viability. When combined with IL-18, it induces apoptosis via the c-Jun N-terminal kinase signalling pathway. Alone, IL-12 appears less cytotoxic. IL-12 skews the phenotype toward both central and effector memory. When added to already expanded γδT cells, upregulates granzyme B and perforin expression. It also promotes secretion of perforin, indicating heightened degranulation and effector activity. In combination with IL-18, IL-12 upregulates inhibitory and exhaustion-associated markers such as CTLA-4, PD-1, T cell immunoglobulin and mucin-domain-containing-3, and Lymphocyte activation gene-3. Induces robust production of proinflammatory mediators, including CCL4, GM-CSF, IL-1Ra, IL-12 (autocrine loop), IL-13, TNF-α, IFN-γ, and IFN-α	10-25 ng/mL	Assy et al[31], García et al[33], Song et al[35]
IL-18	+/-	Promotes expansion of γδT cells by mitigating zoledronate-induced toxicity, indirectly supporting proliferation. Drives the differentiation toward an effector memory phenotype. Enhances cell survival by increasing anti-apoptotic signalling pathways. Upregulates expression of protein kinase B, Bcl-2, phosphorylated I kappa B, and cellular Fas-associated death domain-like interleukin-1 beta-converting enzyme-like inhibitory protein, while downregulating caspase 3 Levels. The effects on cytotoxicity are somewhat contradictory. While some data report downregulation of granzyme B and IFN-γ at the expression level, IL-18 also promotes the generation of CD56brightCD11c+ γδT cells with high cytotoxic cytokine release, co-expressing CD25, CD122, NKG2D, and HLA-DR. Enhances secretion of IFN-γ, TNF-α, and GM-CSF, while not inducing IL-12 production	10-200 ng/mL	Song et al[35], Sugie et al[36], Li et al[37], Nussbaumer et al[41], Tsuda et al[58]
IL-23	+/-	No conclusive data indicate a direct effect of IL-23 on γδT cell proliferation. Enhances the expression of IL-23R mRNA. Induces elevated IL-17 and IFN-γ expression. Combined with CD26 Ligation, promotes the generation of CD26-negative cytotoxic γδT cells. Enhances CD94, CD226, and granzyme B expression. However, the effect is noted only on CD26-positive cells	10-50 ng/mL	Guggino et al[11], Capietto et al[46]
IL-27	+	No conclusive data indicate a direct effect of IL-27 on γδT cell proliferation. Enhances cytotoxicity specifically in resting γδT cells, increasing the expression of granzyme A, granzyme B, and perforin. These effects are not observed in already activated or expanded γδT cells. Upregulates CD62 L, suggesting a shift toward a central memory phenotype. It does not affect the expression of CD16 or NKG2A. In activated γδT cells, IL-27 does not affect the secretion of IL-2, IL-4, IL-17A, IFN-γ, IL-10, IL-1β, or TNF-α. In contrast, in resting γδT cells, IL-27 enhances IL-17A secretion. Additionally, it leads to downregulation of Th2-type cytokines	100 ng/mL	Morandi et al[49]
IL-33	+	Promote in vivo proliferation in dose dose-dependent manner, requiring CD4 T lymphocytes. Exhibit an effector memory phenotype (CD27-, CD45RA-). Elevated cytotoxic potential has been observed, characterised by increased granzyme B, perforin, and CD107a expression. Upregulation of CCR5, CXCR3, NKG2D, CD161, and TNF-related apoptosis-inducing ligand. It does not affect the expression of CD16, CD28, or NKG2S. Moreover, KIR2DL-1, KIR2DL-2, and KIR2DL-3 are not expressed. Promotes secretion of Th1-type cytokines.	100-1000 ng/mL	Duault et al[13]
TGF-β	+/-	Shows contradictory effects on γδT cell expansion. In some settings, it supports robust proliferation of purified Vδ2 T cells. However, when added to already expanded γδT cells, it may inhibit proliferation and delay maturation. Promotes differentiation toward central memory (CD27+, CD45RA-) and effector memory (CD27-, CD45RA+) phenotypes. It also supports the emergence of regulatory-like FoxP3+ γδT cells, especially when combined with IL-15. The impact of TGF-β on cytotoxicity is context-dependent. In some studies, it enhances cytotoxic potential and conjugate formation with tumor cells, associated with CD103 upregulation. However, downregulation of CD107a, absence of intracellular granzyme B, perforin, and IFN-γ, and reduced NKG2D expression have also been observed. Upregulates CCR4, CCR7, CXCR3, CXCR4, LFA-1, CD56, and CD103, while downregulating CCR6, CCR10, CD107a, and NKG2D. It also induces expression of FoxP3, CD25, CD69, CTLA-4, HLA-DR, and CD45RA in certain conditions, particularly in combination with IL-15. Promotes secretion of IFN-γ, TNF-α, IL-9, and granzyme B. However, granzyme A expression is reduced, and perforin levels remain comparable to the standard IL-2 + zoledronate protocol	1.7-10 ng/mL	Peters et al[43], Casetti et al[45], Capietto et al[46]
IFN-γ	0	No specific data on proliferation effects have been reported	1-100 ng/mL	Vermijlen et al[26]

CCL: C-C motif ligand:; CCR9: C-C chemokine receptor 9; CXCL: C-X-C motif ligand; FoxP3: Forkhead box protein 3; GM-CSF: Granulocyte-macrophage colony-stimulating factor; HLA: Human leukocyte antigen; ICAM-1: Intercellular adhesion molecule-1; IFN-γ: Interferon-gamma; IL: Interleukin; KIR2DL: Killer cell Ig-like receptors; KLRG1: Killer cell lectin-like receptor subfamily G, member 1; LFA-1: Lymphocyte function-associated antigen-1; NKG: Natural killer group; PD-1: Programmed cell death protein 1; TGF-β: Transforming growth factor-beta; Th: T helper; TNF: Tumor necrosis factor; +: Positive; -: Negative; +/-: Ambivalent; 0: No evidence of a significant effect for cancer therapies.

Table 2 Selected phosphoantigens used for in vitro activation or expansion of Vδ2 cells

Abbrev name	Full name	Relative strength	Source	Stability in aqueous solution	Ref.
IPP	Isopentenyl pyrophosphate	Approximately 1000 × weaker than HMBPP	Eucaryotic cells	Stable	Latha et al[63], Vantourout et al[64]
HMBPP	(E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate	Most potent	Prokaryotic cells	Not very stable	Kilcollins et al[65]
BrHPP	Bromohydrin pyrophosphate	Stronger than isopentenyl pyrophosphate, weaker than HMBPP	Synthetic	Stable	Sicard et al[66]
MEP	Monoethyl phosphate	Unknown	Prokaryotic cells	Stable	Tanaka et al[67]

HMBPP: (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate.

Table 3 Comparison of different N-aminobisphosphonates

Name	Food and Drug Administration approval	Water solubility	Relative potency1	Ref.
Zoledronate	Yes	4.17 mg/mL	10000 ×	Kondo et al[68], Ruggiero et al[69]
Pamidronate	Yes	50 mg/mL	100 ×	Ruggiero et al[69], Kunzmann et al[70]
Alendronate	Yes	20 mg/mL	1000 ×	Ruggiero et al[69]
Ibandronate	Yes	72 mg/mL	5000 ×	Ruggiero et al[69]
Risedronate	Yes	11 mg/mL	5000 ×	Ruggiero et al[69]
Minodronate	No	20 mg/mL2	10000 ×	Ohishi and Matsuyama[71]
Neridronate	No	5 mg/mL2	100 ×	Ruggiero et al[69]
Olpadronate	No	50 mg/mL2	1000 ×	Ruggiero et al[69]

¹Relative potency with etidronate (a first-generation bisphosphonate) as the reference compound.

²Special conditions may be needed for water solubility, e.g., ultrasonic treatment, heating, pH adjustment.