Non-Vγ9Vδ2 T Cell Activation and Expansion

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γδ T-cell clones could be generated through co-culture of single γδ T cells with artificial antigen-presenting cells (aAPC), and this can facilitate studies to determine Vδ/Vγ pairing, corresponding TCRγδ ligands, and pathogenic reactivity. As a global-leading CRO company, Creative Biolabs has established an advanced platform to provide comprehensive non-Vγ9Vδ2 T cell activation and expansion services.

Why Non-Vγ9Vδ2 Cells Expansion is Needed?

Populations of γδ T cells outside of the Vγ9Vδ2 subset have been grown with immobilized TCRγδ agonists. Plate-bound recombinant MICA and IL-2 were used to sustain the proliferation of γδ T-cell cultures ex vivo from epithelial ovarian cancer and CRC tumor-infiltrating lymphocytes (TILs) and resulted in high frequencies of Vδ1 cells. In addition, plate-bound pan-TCR γδ-specific antibody and IL-2 led to proliferation of both Vδ2 and Vδ1 cells (Vδ2 >>Vδ1) from peripheral blood-derived from both healthy donors and patients with lung cancer or lymphoma. Similarly, OKT3 has been used in combination with IL-2 and IL-4 to stimulate CD4/CD8-depleted T cells from healthy peripheral blood, which resulted in the expansion of Vδ2 and Vδ1 cells (Vδ2 >Vδ1), albeit with reduced cell numbers compared to the TCRγδ monoclonal antibody (mAb)- stimulated cells. A more complex cocktail of cytokines [IL-2, IL-12, and Interferon-γ (IFNγ)] has also been used with OKT3 and CD2-specific antibodies to expand γδ T cells, but the Vδ repertoires were not reported. The transition of these immobilized antigens and antibodies into clinical manufacture will streamline the application of these expansion strategies for γδ T cells and could be the source of clinical trials with non-Vγ9Vδ2 cells.

Activation of γδ T cells purified from PBMCs or cord blood with aAPCs induces clinical-grade expansion of polyclonal γδ T-cell lines with broad specificity toward cancer cells. Fig. 1 Summary of γδ T-cell functions and the roles of specific subsets. (Rajoriya, 2014)

Approaches for Non-Vγ9Vδ2 cells Expansion

Highly polyclonal γδ T cells have been generated through coculture of the patient or healthy donor γδ T cells with irradiated artificial antigen-presenting cells (aAPC), IL-2, and IL-21. The aAPC are derived from the chronic myelogenous leukemia (CML) cell line K562 following genetic modification with T-cell co-stimulatory molecules (CD86 and CD137L), Fc receptors for antibody loading (introduced CD64 and endogenous CD32), antigens (CD19), and cytokines (a membrane-bound IL15), and have been produced as a master cell bank (MCB). γ-irradiation of aAPC before co-culture with T cells subjects the aAPC to death (typically at or within 3 days) thereby reducing the risk for unintended transfer of this tumor cell line into recipients.

Isolation of Non-Vγ9Vδ2 Cells

  • Unlabeled/negative magnetic bead selection
  • Recent reports demonstrated that circulating γδ T cells, containing a polyclonal TCRγδ repertoire, could be isolated from healthy donor venipuncture or umbilical cord blood by unlabeled/negative magnetic bead selection and recursively stimulated with irradiated aAPC, IL-2, and IL-21. The aAPC-expanded γδ T cells proliferated to numbers sufficient for clinical use while maintaining the expression of most TRDV and TRGV alleles and demonstrating TCRδ surface expression of Vδ1 >Vδ1negVδ2neg >Vδ2. These polyclonal γδ T-cell cultures displayed broad tumor reactivity as they were able to lyse leukemia, ovarian cancer, pancreatic cancer, and colon cancer cells. Separation of the polyclonal cultures by TCRδ surface expression showed that each T-cell subset had anti-tumor reactivity and that a polyclonal γδ T-cell population led to the superior survival of mice with established ovarian cancer xenografts.

  • Positive/labeled microbeads selection
  • Another approach isolated polyclonal γδ T cells from PBMC of healthy donors or patients with neuroblastoma by first depleting monocytes followed by positive/labeled selection with anti-TCRγδ-hapten antibody and anti-hapten microbeads. This study made use of the Fc receptors on the aAPC surface to load antiTCRγδ antibody where isolated γδ T cells were co-cultured with the antibody-loaded aAPC. These expanded γδ T cells expressed multiple TRDV and TRGV alleles with surface TCRδ expression of Vδ2 >Vδ1 >Vδ1negVδ2neg. Using this mode of expansion, Vδ1 and Vδ2 were mediators of antibody-independent (AIC) and antibody-dependent cellular cytotoxicity (ADCC), respectively, to neuroblastoma tumor cells (as predicted by whether or not they expressed Fc receptor CD16).

Services at Creative Biolabs

aAPC-expanded polyclonal γδ T cells could be used for anti-tumor therapies because aAPC is currently available as a clinical reagent. In recent years, non-Vγ9Vδ2 cell expansion has shown its significance. Focus on cellular immunotherapies for years, Creative Biolabs proposes comprehensive research services for the development of non-Vγ9Vδ2 cell activation and expansion. Our scientists specialized in immunotherapy also provide global customers with high-quality customized services. Please feel free to contact us for your exclusive solution.


  1. Déchanet-Merville, J. Promising cell-based immunotherapy using gamma delta T cells: Together is better. 2014, 20(22): 5573-5575.
  2. Deniger, D. C.; et al. Clinical applications of gamma delta t cells with multivalent immunity. Front Immunol. 2014, 5: 636.
  3. Rajoriya, N.; et al. Gamma Delta T-lymphocytes in Hepatitis C and Chronic Liver Disease. Frontiers in Immunology. 2014, 5(12): 400.
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