In Vitro Expansion of Vδ1+ T Cells from Cord Blood by Using Artificial Antigen-Presenting Cells and Anti-CD3 Antibody

Abstract

γδ T cells have the potential for adoptive immunotherapy since they respond to bacteria, viruses, and tumors. However, these cells represent a small fraction of the peripheral T-cell pool and require activation and proliferation for clinical benefits. In cord blood, there are some γδ T cells, which exhibit a naïve phenotype, and mostly include Vδ1+ T cells. In this study, we investigated the effect of CD3 signaling on cord blood γδ T-cell proliferation using K562-based artificial antigen presenting cells expressing costimulatory molecules. There were significantly more Vδ1+ T cells in the group stimulated with anti-CD3 antibody than in the group without. In cultured Vδ1+ T cells, DNAM-1 and NKG2D were highly expressed, but NKp30 and NKp44 showed low expression. Among various target cells, Vδ1+ T cells showed the highest cytotoxicity against U937 cells, but Daudi and Raji cells were not susceptible to Vδ1+ T cells. The major cytokines secreted by Vδ1+ T cells responding to U937 cells were Granzyme B, IFN-γ, and sFasL. Cytotoxicity by Vδ1+ T cells correlated with the expression level of PVR and Nectin of DNAM-1 ligands on the surface of target cells. Compared to Vδ2+ T cells in peripheral blood, cord blood Vδ1+ T cells showed varying cytotoxicity patterns depending on the target cells. Here, we determined the ideal conditions for culturing cord blood Vδ1+ T cells by observing that Vδ1+ T cells were more sensitive to CD3 signals than other subtypes of γδ T cells in cord blood. Cultured cord blood Vδ1+ T cells recognized target cells through activating receptors and secreted numerous cytotoxic cytokines. These results are useful for the development of tumor immunotherapy based on γδ T cells.

Keywords: PBMC; Vδ1+ T cells; anti-CD3 antibody; artificial antigen presenting cells; cord blood; γδ T cells.

Figure 1

Effects of aAPCs with anti-CD3 antibody on proliferation of γδ T cells negatively isolated from cord blood. (a) Distribution of Vδ1, Vδ2, and Vδ1-Vδ2- γδ T cells after negative isolation with magnetic beads from cord blood samples (n = 31). (b) Increased γδ T-cell proliferation as shown with an anti-CD3 antibody (n = 5). (c) Distribution of Vδ1+, Vδ2+, and Vδ1-Vδ2- γδ T cells according to the presence or absence of anti-CD3 antibody in culture for two weeks (n = 4). Data shown in (b,c) are the mean ± SD of Vδ1+ T cell frequencies from 4 independent cord blood donors. p values were calculated using Wilcoxon matched-pairs signed rank test. ns: not significant, aAPCs: artificial antigen presenting cells.

Figure 2

Selective expansion of Vδ1+ T cells from cord blood. (a) Flow cytometric analysis of Vδ1+ T cells before and after FACS sorting on the 7th day of culture and after 14 days of culture. (b) Proportion of Vδ1+ T cells before and after sorting on day 7 and on day 14 (n = 7). (c) Fold expansion of Vδ1+ T cells sorted on the 7th day (n = 7). Data shown in (a) are representative of Vd T cell frequencies from one cord blood donor. Data shown in (b,c) are the mean ± SD of Vδ1+ T cell frequencies from 7 independent cord blood donors. p values were calculated using Wilcoxon matched-pairs signed rank test. ns: not significant.

Figure 3

Immunologic memory and cytolytic markers on Vδ1+ T cells. (a) Representative flow cytometry plots showing CD27 and CD45RA expression with gates indicating Naïve (CD45RA+CD27+), central memory (TCM, CD45RA-CD27+), effector memory (TEM, CD45RA-CD27−), and CD45RA+ terminal effector memory cells (TEMRA, CD45RA+CD27−) Vδ1+ T cells on day 14. Frequency of TEMRA, effector memory, central memory, and naïve Vδ1+ T cells shown as a percentage of all Vδ1+ T cells (n = 5). (b) Expression of activating receptors and (c) inhibitory molecules on Vδ1+ T cells (n = 8).

Figure 4

Expression of activating receptor ligands in target cells and Vδ1+ T-cell cytotoxicity. (a) Cytotoxic assays using cultured Vδ1+ T cells were performed against U937, K562, Daudi, and Raji cells in vitro (n = 6). (b) Expression of activating receptor ligands on target cell lines analyzed by flow cytometry and various E:T ratios (from 20:1 to 1.25:1). γδ T cells were co-cultured with calcein AM-labeled target cells at the indicated E:T ratios for 4 h. The supernatants were harvested, and fluorescence analyzed with a microplate reader. The percentage of specific lysis was calculated as [(experimental emission − spontaneous emission) / (maximum emission − spontaneous emission)] × 100. Data shown in (a) are the mean ± SD of % lysis of indicated tumor by Vδ1+ T cells co-cultured in 3 technical replicates for the indicated E:T ratios and from 6 independent donors.

Figure 5

Vδ1+ T-cell secretome when co-cultured with tumor target cells. Vδ1+ T cells were co-cultured with or without target cell lines at an E:T ratio of 2:1 for 24 h. The supernatants were harvested, and cytokine profiles analyzed using a LEGENDplex human CD8/NK panel according to the manufacturer’s protocol. Experiments were analyzed with one-way ANOVA (n = 7). Data shown are the mean ± SD of concentration of molecules or cytokines secreted by Vδ1+ T cells from 7 cord blood donors cultured alone or with indicated tumor cells, and representative of 2 independent experiments. (* p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.00005). E:T, effector:target; SD, standard deviation.

Figure 6

Comparison of cytotoxic effects between cord blood Vδ1+ and adult Vδ2+ T cells. For the cytotoxicity assay, Vδ1+ T cells from cord blood (n = 3) and Vδ2+ γδ T cells from PBMC (n = 3) were co-cultured with calcein AM-labeled U937, K562, and Raji cells at the indicated E:T ratios for 4 h. Supernatants were harvested, and fluorescence analyzed with a microplate reader. The percentage of specific release was calculated as [(Experimental Release − Spontaneous Release) / (Maximum Release − Spontaneous Release)] × 100. Graphs show mean ± SD of data from three independent donors. Experiments were performed in triplicate using two-way ANOVA. ns: not significant.