1Department of Immunotherapeutics (Medinet), University of Tokyo Hospital, 2MEDINET Co., Ltd
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Kondo, M., Izumi, T., Fujieda, N., Kondo, A., Morishita, T., Matsushita, H., et al. Expansion of Human Peripheral Blood γδ T Cells using Zoledronate. J. Vis. Exp. (55), e3182, doi:10.3791/3182 (2011).
Human γδ T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of γδ T cells in peripheral blood have the Vγ9Vδ2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, γδ T cells are attractive candidate effector cells for cancer immunotherapy. Vγ9Vδ2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of γδ T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and γδ T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to γδ T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating γδ T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate Vγ9Vδ2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of γδ T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of γδ T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of γδ T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded γδ T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-γ production assay. 7
1. Isolation of PBMC
2. Expansion of γδ T cells
3. Phenotypic analysis by flow cytometry
4. IFN-γ production assay10
5. Representative Results:
It is important to determine the percentage of γδ T cells in PBMC at the initiation of culture. As shown in Fig. 2 A, the percentage of CD3+TCRVγ9+ γδ T cells in PBMC was 1.6% on day 0. The dominant populations were CD27+CD45RA+ naive or CD27+CD45RA- central memory phenotypes. When γδ T cells were efficiently stimulated, they formed clusters on days 3-5 (Fig. 3 A and B). When cluster formation was delayed, the growth of other cell types, such as CD4+ or CD8+ αβ T cells or NK cells could dominate the growth of γδ T cells (Fig. 3 C and D). After 14 days of culture, the frequency of γδ T cells increased to more than 93.8% of the cultured cells in successful γδ T cell cultures (Fig. 2 E). The cultured γδ T cells upregulated NKG2D and CD69 expression (Fig. 2 G and H). They displayed CD27-CD45RA- effector memory phenotype (Fig. 2 F). The functions of γδ T cells were evaluated with regard to cytokine production and cytotoxicity. The intracellular IFN-γ staining demonstrated that γδ T cells produced IFN-γ in response to PMA/ionomycin treatment or Z-Daudi cells that accumulated IPP after zoledronate treatment (Fig. 4). These results indicate that zoledronate can efficiently stimulate and expand functional γδ T cells.
|7||TCR Vγ9||mouse IgG1|
|9||TCR Vγ9||mouse IgG1||mouse IgG1|
Table 1. Monoclonal antibodies used in multicolor staining of γδ T cells. An example of the phenotypic analysis of γδ T cells performed in our laboratory is shown in Fig. 2.
Figure 1. Separation of PBMC. Blood (7.5-8.0 ml) is drawn into a BD Vacutainer CPT Cell Preparation Tube with Sodium Heparin and directly centrifuged for 20 min at 1800 x g. After centrifugation, the resulting layers as seen from top to bottom: a) Plasma - b) PBMC and platelets - c) Density solution - d) Polyester gel - e) Granulocytes - f) Red blood cells.
Figure 2. Typical surface phenotype of γδ T cells. PBMC were stimulated with zoledronate and IL-2 for 14 days. Cells were stained with FITC-labeled anti-TCR Vγ9 and PE/Cy5-labeled anti-CD3 to monitor the expansion of γδ T cells (A and E). γδ T cells were identified by their expression of TCRVγ9, and their expression of CD27 and CD45RA (B and F), NKG2D (C and G), or CD69 (D and H) was examined.
Figure 3. Representative γδ T cell cultures. PBMC were stimulated with IL-2 (1000 IU/ml) and zoledronate (5 μM). Representative fields are shown (IX71 inverted microscope [Olympus] x 200). Clusters and aggregates of γδ T cells can be observed on day 3 (A) and day 5 (B), when γδ T cells were successfully expanded. In contrast, no clusters or aggregates were observed when γδ T cell growth was not adequate (C and D).
Figure 4. IFN-γ production. γδ T cells were incubated with RPMI-10 only (A) or PMA/ionomycin (B) or Z-Daudi (C) for 4 hr. First, surface expression of TCRVγ9 was stained and then IFN-γ production was examined by intracellular IFN-γ staining.
Figure 5. Kinetics of γδ T cell culture. (A) Absolute number of cultured cells, (B) percentage of γδ T cells, and (C) absolute number of γδ T cells at the indicated time points.
The method presented here enables efficient expansion of γδ T cells from PBMC. γδ T cells activated and expanded by zoledronate and IL-2 develop complete effector functions, reflected by cytokine production and cytotoxicity. It has been reported that the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP) and 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP) also expand γδ T cells; however, they are not commercially available. In contrast, zoledronate is already licensed for clinical applications as Zometa. Therefore, a reliable reagent is easily available.
The selection of culture media and serum is critical. Use appropriate culture media such as ALyS203 (Cell Science & Technology Institute) or OpTmizer (Invitrogen) for successful γδ T cell expansion.11 Verify that autologous plasma, pooled human AB sera or FCS can support γδ T cell culture. Also remember that PBMC from some donors fail to respond to zoledronate stimulation regardless of other culture reagents. If that happens, the only option is to change the donor.
As we have demonstrated, enrichment of γδ T cells was achieved relatively early; almost 80 % of cultured cells were γδ T cells by day 7. γδ T cells continued to proliferate up to 12-14 days (Fig. 5). Approximately 2.2 x 108 γδ T cells can be obtained from 1 x 106
PBMC containing 1.6 x 104 γδ T cells. This culture method has been used in phase I clinical trials evaluating the safety and feasibility of Zoledronate-expanded γδ T cell transfer therapy in patients with multiple myeloma or lung cancer.12,13
No conflicts of interest declared.
|PROLEUKIN||Novartis AG||human recombinant IL-2|
|BD Vacutainer CPT Cell Preparation Tube with Sodium Heparin||BD Biosciences||362753|
|ALyS203- medium||Cell Science & Technology Institute||0301-7|
|phorbol 12-myristate 13-acetate (PMA)||Sigma-Aldrich||P1585-1MG|
|IntraPrep||Beckman Coulter Inc.||A07803|
|anti-human CD3-FITC or PE/Cy5||Beckman Coulter Inc.||A07746 FITC A07749 PE/Cy5|
|anti-human CD4-ECD||Beckman Coulter Inc.||6604727|
|anti-human CD8-PE/Cy5||Beckman Coulter Inc.||6607011|
|anti-human CD14-PE/Cy5||Beckman Coulter Inc.||A07765|
|anti-human CD19-PE||Beckman Coulter Inc.||A07769|
|anti-human CD45-ECD||Beckman Coulter Inc.||A07784|
|anti-human CD56-PE/Cy5||Beckman Coulter Inc.||A07789|
|anti-human TCRαβ-PE||Beckman Coulter Inc.||A39499|
|anti-human TCR Vγ9-FITC||Beckman Coulter Inc.||IM1463|
|anti-human CD27-PE/Cy5||Beckman Coulter Inc.||6607107|
|anti-human CD45RA-ECD||Beckman Coulter Inc.||IM2711|
|anti-human CD69-PE||BD Biosciences||555531|
|anti-human NKG2D-PE||Beckman Coulter Inc.||A08934|
|Anti-humal IFNγ-PE||Beckman Coulter Inc.||IM2717U|
|Mouse IgG1 isotype control-PE||Beckman Coulter Inc.||A07796|
|Mouse IgG1 isotype control-ECD or PE/Cy5||Beckman Coulter Inc.||A07797A07798|