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

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.


Introduction
γδ T cells express distinct T-cell receptor (TCR) γ and δ chains on their surface, accounting for 0.5%-5% of all T-lymphocytes in peripheral blood [1]. Particularly, they are more widespread within epithelial tissues where they can comprise up to 50% of T cells [2]. Unlike αβ T cells, these cells are not restricted by the major histocompatibility complex and could respond to antigens without conventional antigen processing [3][4][5][6]. They rapidly increase in patients with infectious diseases and tumors, producing various cytokines according to inflammatory environments [5,[7][8][9]. The Vδ2+ and non-Vδ2+ (mainly Vδ1+) subsets of human γδ T cells have potent antitumor effects due to distinct homing patterns and activating killer receptors [10]. While the TCR of Vδ2+ T cells primarily recognizes small bacterial phosphoantigens, alkylamines, and tumor cell-derived pyrophosphates, Vδ1+ T cells preferentially recognize stress-inducible ligands such as MICA/B, ULBPs, and several other ligands [11,12].

Cytotoxicity Assay
For the cytotoxicity assay using calcein AM (Invitrogen), target cells were stained with 5 µM calcein AM and incubated for 20 min at 37 • C and 5% CO 2 in the dark and washed twice with phosphate-buffered saline and suspended in 10% RPMI at 1 × 105 cells/mL. Effector and target cells were co-cultured at the indicated effector:target ratios in a 96-well U-bottom plate. Triton X-100 was added to a final concentration of 2% for maximum fluorescence release and only target cells were cultured without effector cells for spontaneous release. After a 4 h incubation, plates were centrifuged at 2500 rpm for 5 min, and the

Secretome Analysis
For secretome analysis, Vδ1+ γδ T cells and Daudi, U937 cells were co-cultured in a 96-well U-bottom plate at a 2:1 ratio for 4 or 24 h; plates were centrifuged at 2500 rpm for 5 min, and the supernatants were carefully transferred to 5 mL round tubes. The supernatants were analyzed using a LEGENDplex human CD8/NK panel (BioLegend) according to the manufacturer's protocol.

Statistics
Statistical significance was determined by paired t-tests, and one-way and two-way analysis of variance. All analyses were performed using Prism 5.01 software (GraphPad Software, San Diego, CA, USA).

Effect of Anti-CD3 Antibody Stimulation on γδ T-Cell Subtypes' Proliferation
To investigate the effect of CD3 signals on the γδ T-cell subtypes of cord blood, γδ T cells negatively isolated with magnetic beads were stimulated for two weeks using irradiated K562-based aAPCs expressing costimulatory molecules, in the presence or absence of anti-CD3 antibody, respectively. The subtypes of γδ T cells negatively isolated from 31 cord blood samples were 11.2% ± 12.5% of Vδ1+ γδ T cells, 2.6% ± 2.3% of Vδ2+ γδ T cells, and 86.1% ± 14% of Vδ1-Vδ2-γδ T cells (Figure 1a). A representative plot of distribution of Vδ1, Vδ2, and Vδ1-Vδ2-γδ T cells after negative isolation with magnetic beads from cord blood samples is presented in Figure S1. On day 14, the fold expansion of the group stimulated with anti-CD3 antibody (328.6 ± 226.6) was significantly higher than that of the group without anti-CD3 antibody (23.0 ± 22.5; Figure 1b). In comparison, the proportion of Vδ1+ T cells increased in the group with anti-CD3 antibody than in that without (52.3% ± 5% vs. 29.9% ± 11.9%; Figure 1c). A representative plot of distribution of Vδ1+, Vδ2+, and Vδ1-Vδ2-γδ T cells is presented in Figure S3. The proportions of Vδ2+ T cells and Vδ1-δ2-T cells showed no significant differences between groups. These data suggest that CD3 stimulation is required for γδ T-cell proliferation in cord blood, and that Vδ1+ T cells are the most sensitive to this type of stimulation. Therefore, in the subsequent experiment, we established a culture method to expand pure Vδ1+ T cells.

Characteristics of Selectively Expanded Vδ1+ T Cells
The isolation and culture of pure Vδ1+ T cells isolated directly from cord blood were not efficient due to low cell recovery and viability. Therefore, after culturing negatively isolated γδ T cells with aAPCs and anti-CD3 antibody for one week, Vδ1+ T cells were separated using a FACS cell sorter and cultured for one more week. The proportions of Vδ1+ T cells before and after sorting on day 7 were 12.2% ± 9.4% and 90.6% ± 8%, respectively. After one week of additional culture, Vδ1+ T cells accounted for 93.2% ± 4.4% of all cells (Figure 2a,b). The fold expansion of purely isolated Vδ1+ T cells between days 7 and 14 was 15.97 ± 8 (Figure 2c).

Effector Functions of Vδ1+ T Cells
Next, we measured the cytotoxicity of Vδ1+ T cells against various tumor cells and the association with the surface ligands of these target cells. Among U937, K562, Daudi, and Raji cells, U937 cells showed the highest susceptibility to killing by Vδ1+ T cells, whereas K562 cells showed moderate susceptibility. However, Daudi and Raji cells were not susceptible to Vδ1+ T cells. For an E:T ratio of 10:1, the lysis percentage for each target cell was 92% ± 11% for U937 cells, 31.3% ± 23.7% for K562 cells, 4.9% ± 5.3% for Daudi cells, and 1.1% ± 4.4% for Raji cells. To determine the reason for the differing lysis degree among cells, we measured the expression of activating receptor ligands in target cells. In U937 cells, most ligands except MICA were highly expressed, and in K562 cells Nectin and B7-H6 were highly expressed. However, Raji cells showed moderate B7-H6 expression and Daudi cells did not express any ligands. Correlation analysis between the expression rates  Figure S5). These results suggest that Vδ1+ T-cell cytotoxicity is affected by the expression levels of activating receptor ligands on the surface of target cells (Figure 4a,b). Representative flow cytometry data of target cell ligands expression is shown in Figure S6. In addition, the levels of activating receptors and inhibitory molecules expressed on the surface of Vδ1+ T cells were analyzed using flow cytometry (n = 8). Among the activating receptors, DNAM-1 (97.7% ± 2.1%) and NKG2D (53.3% ± 11.1%) showed high ex-  expression rates of each ligand and the cytotoxicity at 10:1 in target cells showed icant correlation with the expression of PVR (r = 0.95, p = 0.041), and with PVR and of DNAM-1 ligands (r = 0.96, p = 0.032) ( Figure S5). These results suggest that Vδ cytotoxicity is affected by the expression levels of activating receptor ligands on face of target cells (Figure 4a,b). Representative flow cytometry data of target cell expression is shown in Figure S6. After co-culturing Vδ1+ T cells and target cells at an E:T ratio of 2:1 for 24 h natants were harvested, and the molecules and cytokines secreted by Vδ1+ T ce After co-culturing Vδ1+ T cells and target cells at an E:T ratio of 2:1 for 24 h, supernatants were harvested, and the molecules and cytokines secreted by Vδ1+ T cells were Vaccines 2023, 11, 406 9 of 14 measured by a bead array. When co-culturing Vδ1+ T and U937 cells, the concentrations of Granzyme B, IFN-γ, and sFasL were significantly higher than those in Daudi cells ( Figure 5). , 11, x FOR PEER REVIEW 9 of 14 measured by a bead array. When co-culturing Vδ1+ T and U937 cells, the concentrations of Granzyme B, IFN-γ, and sFasL were significantly higher than those in Daudi cells (Figure 5). Additionally, we compared the cytotoxicity of cord blood Vδ1+ T cells and adult Vδ2+ T cells to U937, K562, and Daudi cells (Figure 6). Vδ2+ T cells were mainly effector memory (73.0 ± 23.3%), and NKG2D (99.6 ± 0.3%) and DNAM-1 (95.8 ± 6.4%) were highly expressed [29]. U937 cells showed significantly higher susceptibility to killing by cord blood Vδ1+ T Figure 5. Vδ1+ T-cell secretome when co-cultured with tumor target cells. Vδ1+ T cells were cocultured 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. Additionally, we compared the cytotoxicity of cord blood Vδ1+ T cells and adult Vδ2+ T cells to U937, K562, and Daudi cells ( Figure 6). Vδ2+ T cells were mainly effector memory (73.0 ± 23.3%), and NKG2D (99.6 ± 0.3%) and DNAM-1 (95.8 ± 6.4%) were highly expressed [29]. U937 cells showed significantly higher susceptibility to killing by cord blood Vδ1+ T cells (77.7 ± 10.54%, E:T ratio = 10:1) than adult Vδ2+ T cells (63.8% ± 12.8%, E:T ratio = 10:1). Meanwhile, K562 cells showed lower susceptibility to killing by cord blood Vδ1+ T cells (49.5% ± 17.4%, E:T ratio = 10:1) than adult Vδ2+ T cells (57.2% ± 34%, E:T ratio = 10:1), and Daudi cells showed susceptibility only to adult Vδ2+ T cells (87.5% ± 11.6%, 6% ± 6.9%, E:T ratio = 10:1). These results suggest a different cytotoxicity mechanism of cord blood Vδ1+ T cells to that of adult Vδ2+ T cells.

Discussion
Although αβ and γδ TCRs both require the CD3 complex for signaling and show similar structures, these receptors had different characteristics in terms of complex geometry, glycosylation pattern, plasma membrane organization, and the accessibility of signaling motifs in the CD3 intracellular tails [32]. In the αβ TCR, an antigen induced conformational change at CD3 subunits is required for αβ TCR activation but not in the human Vδ2+ TCR [33]. Stimulation of CD4/CD8-depleted T cells with OKT3 and γδ TCR monoclonal antibody resulted in the expansion of Vδ2+ T and Vδ1+ T cells (Vδ2 > Vδ1) [13,34]. In this study, we cultured cord blood γδ T cells using aAPCs with or without anti-CD3 antibody, and observed that γδ T cells cultured with anti-CD3 antibody showed higher proliferation than γδ T cells cultured without anti-CD3 antibody; particularly, Vδ1+ T cells proliferated more than Vδ2+ T cells (Figure 1). Stimulation with aAPCs expressing various costimulatory molecules and anti-CD3 antibody may inhibit the apoptosis triggered by TCR/CD3 signaling, inducing proliferation [35]. 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.

Discussion
Although αβ and γδ TCRs both require the CD3 complex for signaling and show similar structures, these receptors had different characteristics in terms of complex geometry, glycosylation pattern, plasma membrane organization, and the accessibility of signaling motifs in the CD3 intracellular tails [32]. In the αβ TCR, an antigen induced conformational change at CD3 subunits is required for αβ TCR activation but not in the human Vδ2+ TCR [33]. Stimulation of CD4/CD8-depleted T cells with OKT3 and γδ TCR monoclonal antibody resulted in the expansion of Vδ2+ T and Vδ1+ T cells (Vδ2 > Vδ1) [13,34]. In this study, we cultured cord blood γδ T cells using aAPCs with or without anti-CD3 antibody, and observed that γδ T cells cultured with anti-CD3 antibody showed higher proliferation than γδ T cells cultured without anti-CD3 antibody; particularly, Vδ1+ T cells proliferated more than Vδ2+ T cells (Figure 1). Stimulation with aAPCs expressing various costimulatory molecules and anti-CD3 antibody may inhibit the apoptosis triggered by TCR/CD3 signaling, inducing proliferation [35].
Cord blood Vδ1+ T cells showed high cytotoxicity in K562 and U937 cells, but not in Raji or Daudi cells. The anti-γδ TCR antibody could expand both the Vδ1 and Vδ2 subsets of peripheral blood γδ T cells. In the comparison of the cytotoxic activity between them, purified Vδ1 and Vδ2 subsets exhibited similar cytotoxicity against Daudi and K562 cells [16]. However, a Vδ1+ T cell line established from human peripheral blood by immortalization with Herpesvirus saimiri was able to specifically recognize K562 cells, but Daudi cells were resistant to Vδ1+ T cell killing [36]. As such, peripheral blood-derived Vδ1+ T cells show different degrees of cytotoxicity to Daudi cells depending on the culture conditions, so it is necessary to confirm this in the future using Vδ1+ T cells amplified under the same culture conditions as in this study. DNAM-1 and NKG2D receptor expression was noted in the Vδ1+ T cells cultured in this study; the expression of PVR, Nectin, ULBPs, and MICA was very low in Raji and Daudi cells compared to K562 or U937 cells. Although B7-H6, an NKp30 ligand, was also highly expressed in K562 and U937 cells, NKp30 expression was very low in cultured Vδ1+ T cells; therefore, it does not seem to play a major role in this type of cytotoxicity. A clinical-grade protocol for expansion and differentiation of "Delta One T" (DOT) cells as peripheral blood Vδ1+ T cells consists of two-steps. In the 1st step, in addition to anti-CD3 antibody, IL-4, IFN-r, IL-21, IL-1b, and in the 2nd step, a combination of cytokines such as IL-15 and IFN-r was used, and these cytokines increased the proliferation of Vδ1+ T cells and the expression of NKp30 [17]. These DOT cells expressed the natural cytotoxicity receptors, NKp30 and NKp44, which synergized with the T-cell receptor to mediate leukemia cell targeting in vitro. DOT cells showed highly cytotoxic activity against acute myeloid leukemia (AML) primary samples and cell lines. AML reactivity was only slightly impaired upon Vδ1+ TCR antibody blockade, whereas it was strongly dependent on expression of the NKp30 ligand, B7-H6 [37]. Peripheral blood Vδ1+ T cells cultured in the presence or absence of IL-2, soluble anti-CD3 antibody (clone OKT3), and IL-15, showed the highest NKp30 expression (28%) in Vδ1+ T cells cultured with the OKT3/IL-2/IL-15 combination [38]. However, cord blood Vδ1+ T cells showed very low NKp30 and NKp44 expression, which is presumed to be due to the culture using only anti-CD3 antibody and IL-2 in this study. It is necessary to investigate the effects of various cytokines in detail in the future. Since we only used IL-2 in this study, we need to investigate the effects of various cytokines, including IL-15, on the expression of NKp30 or NKp44 receptors [38].
Daudi cells showed the greatest difference in cytotoxicity of cord blood Vδ1+ T cells proliferated compared to adult peripheral blood Vδ2+ T cells. Vδ2+ T cells are activated by cells that accumulate HMBPP and/or IPP and can also be stimulated through receptors NKG2D and DNAM-1 of various stress-induced molecules expressed on tumor cells including MICA/B, UL16-binding proteins (ULBP1-6), Nectin-2, and PVR, respectively [39,40]. Daudi cells generate endogenous phosphorylated metabolites by upregulating the mevalonate pathway, which results in intracellular IPP accumulation [39,41]. The expansion and pattern of responses of multiple Vδ2+ T cells to Daudi cells was similar to that of IPP stimulation [42]. Since PVR, Nectin, ULBPs, MICA, and B7-H6, ligands of the activating receptor of Vδ1+ T cells, were barely expressed in Daudi cells, no Vδ1+ T-cell cytotoxicity was observed. The cytotoxicity of Vδ1+ T cells to U937 cells was significantly higher than that of Vδ2+ T cells; this is probably due to the high expression of the active receptor in Vδ1+ T cells, or differences in the secretion ability of cytokines associated with cytotoxicity ( Figure 6). As a result of a blocking test for these receptors using antibodies against DNAM-1 and NKG2D expressed in Vδ1+ T cells, it was confirmed that some of the cytotoxicity of Vδ1+ T cells against U937 cells was partially reduced ( Figure S7).
In a co-culture of Vδ1+ T cells and U937, the concentrations of Granzyme B, IFN-γ and sFasL were significantly higher than those of Daudi cells ( Figure 5). Perforin and granzymes synergize to mediate target cell apoptosis: pro-apoptotic granzymes diffuse through perforin pores on the plasma membrane of target cells [43]. Vδ1+ T cells showed high cytotoxicity against U937 cells, probably due to the high secretion of both perforin and Granzyme B. However, in a similar test using adult Vδ2+ T cells, IFN-γ, TNF-α, and sFasL, among secreted cytokines after co-culture with Daudi cells, target cells that showed high cytotoxicity were significantly higher than those of U937 with low cytotoxicity [29]. Interestingly, the concentration of Granzyme B secreted from Vδ1+ T cells was about 10 times higher than that of Vδ2+ T cells, while IFN-γ and TNF-α concentrations secreted by Vδ1+ T cells were lower than those of Vδ2+ T cells [29].
In summary, we established the conditions for culturing cord blood Vδ1+ T cells by observing that, among cord blood γδ T cells, Vδ1+ T cells were more sensitive to CD3 signals than other subtypes, and thus effectively proliferated. Cultured cord blood Vδ1+ T cells expressed high DNAM-1 and NKG2D, and showed strong cytotoxicity and high secretion of Granzyme B, sFasL, and IFN-γ by recognizing target cells with their ligands. Although Vδ2+ T cells derived from adult blood were compared with cord blood Vδ1+ T cells in this study, a direct comparison with Vδ1+ T cells derived from adult blood or Vδ1+ derived from other tissue sources is necessary to investigate the biology of human γδ T cells. Since the stimulation of Vδ1+ T cells with aAPC and anti-CD3 antibody used in this study was the same as the αβ T cell stimulation conditions, and the proliferation rate was similar, further study is needed to prove whether the structural properties of the Vδ1 TCR and CD3 complex would have similar structural properties to those of the αβ TCR.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/vaccines11020406/s1, Figure S1: Representative plot of distribution of Vδ1, Vδ2, and Vδ1-Vδ2-γδ T cells after negative isolation with magnetic beads from cord blood samples.; Figure S2: Absolute cell number of Vd1+ T cells presence or absence of anti-CD3 antibody in culture for two weeks. The absolute cell numbers of 5 cordblood units increased. (n = 5).; Figure S3: Representative plot of 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.; Figure S4: Absolute cell number of Vδ1+ T cells with anti-CD3 antibody and aAPCs in culture for two weeks. The absolute cell numbers of 7 cordblood units increased. (n = 7).; Figure S5: Correlation analysis between the expression rate of each ligand.; Figure S6: Representative flow cytometry data of target cell ligands expression.; Figure S7: Result of a blocking test for these receptors using anti-bodies against DNAM-1 and NKG2D expressed in Vd1+ T cells.