Human Vγ9δ2 (Vδ2) T cells represent a unique effector T cell population in humans and primates detecting nonpeptid phosphoantigens, playing an important role in antimicrobial and antitumor immunity. Currently, it is believed that various leukocyte subsets can promote phosphoantigen-driven Vδ2 cell expansion, but the essential cell type required remains elusive. We have used high purity cell sorting to analyze the cellular requirements for (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMBPP)–driven Vδ2 cell expansion. To our knowledge, we show for the first time that primary human MHC-class II+ cells are indispensable for HMBPP- and isopentenylpyrophosphate-driven Vδ2 cell expansion. In contrast, MHC-class II cells are unable to promote Vδ2 cell expansion. Moreover, purified primary human TCRαβ+ T cells, CD4+, or CD8+ T cells also failed to promote HMBPP-mediated Vδ2 expansion. Depletion of CD4+CD25+ T cells demonstrated that inability of TCRαβ+ cells to expand Vδ2 cells was not related to the presence of regulatory T cells. Separation of MHC-class II+ cells into dendritic cells, monocytes, and B cells revealed that dendritic cells were the most potent Vδ2 expanders. Pulsing experiments demonstrated that HMBPP transforms MHC-class II+ but not negative cells into Vδ2 expanders. MHC-class II–blocking experiments with mAbs and secondary MHC-class II induction on CD4+ T cells after CD3/CD28 costimulation indicated that MHC-class II is necessary, but not sufficient to promote Vδ2 expansion. Our results provide novel insight into the primary cell-specific requirements for human Vδ2 expansion.

Human γδ T cells represent only a small fraction of CD3+ T lymphocytes in peripheral blood (0.5–10%). The majority of them express TCR comprising Vγ9 and Vδ2 V regions, hereafter referred to as Vδ2 cells (13). These Vδ2 cells recognize nonpeptidic phosphorylated metabolites of isoprenoid biosynthesis, generally called phosphoantigens (P-Ag), such as the most potent activator (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), isopentenyl pyrophosphate (IPP), and its isomer dimethylallyl pyrophosphate (47). Additionally, aminobisphonates, such as zoledronate and pamidronate (PAM), promote endogenous IPP accumulation via inhibition of farnesyl pyrophosphate synthase in cells and subsequent Vδ2 cell proliferation (8, 9).

Although the molecular mechanisms for the presentation of P-Ag to Vδ2 cells are currently unknown, increasing information suggests the involvement of different recognition and activation mechanisms according to the P-Ag or drug tested. Previous studies with cell lines and clones reported that pyrophosphates do not require uptake and processing (10). In addition, IPP seems to bind to a putative presenting molecule with low affinity because it could not be effectively pulsed on APC (10). However, more recently, photoaffinity HMBPP analogs were efficiently pulsed on tumor cell lines (11), and also HMBPP could stably associate to a putative molecule in the membrane of APC using a Vγ9Vδ2 TCR tetramer construct that was able to bind HMBPP-pulsed APC lines (12). In the same way, several tumor cell lines pulsed with either pamidronate or synthetic pyromonophosphoesters are able to efficiently promote the specific γδ T cell proliferation and IFN-γ production in a TCR-dependent manner (1315).

Regarding the cellular requirements for Vδ2 cells to be activated and expand, it was suggested that clones of γδ T cells can respond to IPP in the absence of accessory APC, and, therefore, this Ag can be presented by γδ T cell clones themselves (10). Wei et al. (12) showed that a Vγ9Vδ2 tetramer stained the cell surface of human macrophages and dendritic cells (DC), and also macaque CD8+ T cells and B cells, suggesting that APC as well as T cells are able to expand Vδ2 cells. On the contrary, activation of primary Vδ2 cells by PAM required the presence of monocyte adherent cells (15). Moreover, immature DC were reported to selectively enhance TNF-α production of synthetic P-Ag bromohydrin pyrophosphate and PAM-stimulated γδ T cells, but not their cytolytic responses (16).

Despite recent progress in understanding Vδ2 cell responses to P-Ag, the specific cellular requirements and recognition modalities of most γδ T cell ligands remain unknown. Although HMBPP is the most potent activator for Vδ2 cells and it is widely distributed among several important human pathogens (4, 17, 18), little is known about the specific cellular requisites for Vδ2 cells to expand in response to HMBPP. Furthermore, majority of previous studies were performed with cell lines and clones. To our knowledge, this is the first study that has used highly purified primary Vδ2 cells and highly purified primary cellular subsets supposed to interact with Vδ2 cells to investigate the specific cellular requirements and conditions for HMBPP-mediated Vδ2 cell expansion. Our results suggest that HMBPP is presented to Vδ2 cells only by MHC-class II+ cells, whereas MHC-class II cells lack this capacity, and, furthermore, this MHC-class II+ cell requirement can be generalized to other pyrophosphates, such as IPP. In addition, Vδ2 cell expansion is greater in the presence of DC than other MHC-class II+ APC, including monocytes and B cells. We also show that HMBPP pulsing transforms MHC-class II+ cells, but not MHC-class II cells, into Vδ2 expanders. Finally, we demonstrate that expression of MHC-class II molecules is necessary to promote Vδ2 cell expansion.

Buffy coats were obtained from randomly selected healthy, voluntary blood donors after giving their informed consent (approved by Ethics Committee, University of Giessen; file 05/00). The following mAb were used in this study: Vδ2-PE (B6 clone), CD3-Pacific Blue (HIT3a clone), αβTCR-Alexa Fluor 647 (IP26 clone), CD4-PE-Cy7 (OKT4 clone), and CD14-allophycocyanin-Cy7 (HCD14 clone) from BioLegend; CD8-allophycocyanin (BW135/80 clone), CD1c (BDCA-1)-allophycocyanin (AD5-8E7 clone), CD45RA-FITC (HI100 clone), CD141 (BDCA-3)- allophycocyanin (AD5-14H12 clone), CD304 (BDCA-4/Neuropilin 1)-allophycocyanin (AD5-17F6 clone), and CD19-FITC (LT19 clone) from Miltenyi Biotec; CD4-PerCP (SK3 clone), HLA-DR-allophycocyanin-H7 (L243 [G46-6] clone), HLA-DR-FITC (L243 [G46-6] clone), and CD25-FITC (OX-39 clone) from BD Pharmingen; and pan-HLA-class II (HLA-DR, DP, DQ, clone Tü39, purified and FITC labeled) and isotype control (clone G155-178, purified and FITC labeled) from BD Biosciences. Daudi (American Type Culture Collection [ATCC]; CCL-213), Raji (ATCC; CCL-86), THP-1 (ATCC; TIB-202), KG-1a (ATCC; CCL 246.1), Meg-01 (ATCC; CRL-2021), and U937 (ATCC; CRL-1593.1) cell lines were provided by S. Santoso (Gießen, Germany).

For each set of experiments, four different buffy coats were screened for Vδ2 cell percentages. To decrease the risk of selecting individuals with pre-expanded or preactivated Vδ2 cells, only donors with Vδ2 cell frequencies <6% and negative infectious serology (HIV, hepatitis B and C, syphilis) were selected for sorting experiments. Screening was performed in total whole blood. Briefly, 200 μL was incubated for 15 min in the dark with Vδ2-PE and CD3-Pacific Blue mAb, and, in some cases, to determine DC numbers, a mixture of the allophycocyanin-labeled CD1c, CD141, and CD304 mAb was also included. After the incubation, the cells were lysed using FACS-lysing solution (BD Biosciences), washed twice, and analyzed by flow cytometry (FACS-Canto II; BD Biosciences).

PBMC were isolated by Ficoll-Paque (GE Healthcare) gradient centrifugation, washed twice, filtered with a 30 μm preseparation filter (Miltenyi Biotec), counted using the Sysmex KX21N (Sysmex, Kobe, Japan), and resuspended in PBS-EDTA-HEPES solution. Fifty to 90 million PBMC were stained with the appropriate quantity of mAb and incubated for 20 min on ice in the dark, washed twice, and filtered again just before the sort. FACS was performed with a BD FACSAria II (BD Biosciences) with a high purity sort mask to ensure maximal purity. Before each experiment, instrument quality control was performed and isotype controls were run. For each cell population, gating strategy for each specific experiment was based on the selection of the target cell subset and exclusion of negative populations in a third channel; for example, using CD3-Pacific Blue, Vδ2-PE, and αβTCR-AlexaFluor 647 mAbs, potential contamination of cells expressing the αβTCR was excluded. Selected cell populations were collected in X-vivo 15 medium (Lonza). After the sort, an aliquot was used to analyze the purity of the isolated populations, which was >99% in all cases (Supplemental Fig. 1).

After the sorting, the cells were washed, counted, and resuspended in the appropriate volume of RPMI 1640 containing stable glutamine (PAA Laboratories), 1 μg/ml gentamicin (PAA Laboratories), 2 mM HEPES (Invitrogen), and 10% pooled human AB serum (Vδ2 cell medium). Cocultures of highly purified Vδ2 cells with the other sorted cell populations or cell lines were performed in 96-round bottom-well plates (Cellstar) at a 1:4 ratio (Vδ2:other population), or, as indicated, during 6 d at 37°C and 5% CO2. In some cases, bulk PBMC were cultured under the same conditions as cocultures. HMBPP was synthesized, as previously described, and used at a concentration of 100 nM (19). IL-2 (PeproTech) was added to the cultures at a concentration of 100 U/ml. In some experiments, IPP (Sigma-Aldrich) was added at 1 μM. After the culture, the cells were collected and washed, and viable cells were counted using trypan blue in a Neubauer chamber. Percentages of Vδ2 cells were analyzed by flow cytometry after blocking for 10 min on ice and staining with the mAb against CD3 and Vδ2, or appropriate isotype controls, for 15 min on ice in the dark. All experiments were performed in duplicate, and every set of experiments was repeated at least three times.

MHC-class II induction in sorted CD4+ T cells was performed after 6-d costimulation with plate-bound CD3 and soluble CD28 mAbs (clones HIT3a and 28.2; 1 μg/ml each; BD Pharmingen). Unstimulated sorted CD4+ T cells, cultivated at low-dose IL-2 (5 U/ml), served as negative controls. MHC-class II expression was determined on day 6 by HLA-DR and pan-HLA-class II mAb surface staining and flow cytometry.

Naive CD8+ T cells were magnetic-bead sorted with the naive CD8+ T cell isolation kit (Miltenyi Biotec) on an automatic magnetic-bead sorter (AutoMacs; Miltenyi Biotec), according to the manufacturer instructions. Sorting purity (>95%) was controlled by CD8 CD45RA surface staining by flow cytometry. FACS-sorted CD1c+ DC were incubated at the indicated ratios in culture medium with CFSE-labeled allogeneic naive CD8+ T cells (1 × 105/well) in 96-well round-bottom wells (Greiner) for 5 d. Culture medium consisted of RPMI 1640 with l-glutamine (PAA Laboratories), penicillin/streptomycin (PAN Biotech), sodium-pyruvate (Life Technologies), nonessential amino acids (Sigma-Aldrich), HEPES buffer (Life Technologies), and 10% heat-inactivated FCS (PAA Laboratories). carboxyfluorescein diacetate succinimidyl ester–labeling concentration was 1 μm, and was performed according to the manufacturer instructions (Vybrant CFDA-Cell Tracer kit; Molecular Probes). Positive CD8+ T cell proliferation controls consisted of CD3/CD28 (clones HIT3a and 28.2; 1 μg/ml each; BD Pharmingen) costimulated cells; negative controls consisted of naive CD8+ T cells cultured in the absence of stimulator cells.

In a first step, MHC-class II+ APC were depleted from PBMC using the HLA-DR-FITC mAb. Lack of potential contamination with DC and CD14+ cells was confirmed (Supplemental Fig. 1). Afterward, DC (mix of allophycocyanin-labeled CD1c, CD141, and CD304 mAb), monocytes (CD14-allophycocyanin-Cy7), and B cells (CD19-FITC) were sorted. For the culture, sorted DC, CD14+, or CD19+ cells were added back to PBMC depleted of MHC-class II+ APC at 10% of the total cell number in the well. The cells were cultured in the presence of 100 nM HMBPP and 100 U/ml IL-2 for 6 d.

MHC-class II+ cells and MHC-class II cells were sorted and incubated with 100 nM HMBPP at either 37°C or 4°C for 1 h. The cells then were washed two times with PBS, and a third time with the Vδ2 cell culture medium described above. Autologous highly purified Vδ2 cells were incubated with the pulsed APC in the presence of 100 U/ml IL-2 for 6 d at a 1:4 ratio. In parallel, HMBPP and IL-2 were also directly added to the coculture as a control for the unpulsed condition. In some experiments after pulsing, MHC-class II+ and MHC-class II cells were fixed with 2% paraformaldehyde for 15 min at room temperature, or lysed by a three-cycle freeze in liquid nitrogen and thaw at 37°C.

Supernatants from cocultures of sorted Vδ2 cells with MHC+ or MHC cells in the presence of HMBPP were collected after 6 d, centrifuged, and stored at −80°C until further use. Human IL-4, IL-6, IL-9, IL-10, IL-15, IL-17, IFN-γ, and TNF-α were quantitated by bioplex protein beads using the Bio-Plex 200 system, according to manufacturer’s instructions (Bio-Rad). Cultures were performed in duplicate and repeated four times, and cytokine measurement was performed in duplicate.

Results are expressed as the mean ± SEM of the Vδ2 cell absolute cell numbers of at least three different experiments. Differences between sample groups were evaluated using the two-tailed nonparametric Mann–Whitney U test. Significant differences were considered for a p value <0.05. Statistic analyses were performed using the Predictive Analytics Software (PASW, 2010).

To establish the optimal time to detect a significant expansion of Vδ2 cells in response to HMBPP without manipulating the culture, PBMC from the same donor were cultured in parallel with 100 nM HMBPP and 100 U/ml IL-2, or without HMBPP and 10 U/ml IL-2. Vδ2 cell expansion started at day 4 in the presence of HMBPP, whereas there was no expansion without HMBPP (Fig. 1A). In addition, to control whether the sorting affected Vδ2 cell expansion capacity, we compared cultures of ex vivo bulk PBMC with sorted PBMC containing mAb. Final Vδ2 cell numbers were comparable between ex vivo cultured PBMC and PBMC under the sorting stress. Therefore, the sorting procedure does not affect Vδ2 cell capacity to expand (Fig. 1B).

FIGURE 1.

Vδ2 cell expansion in response to HMBPP within bulk PBMC. (A) Kinetics of the percentage of Vδ2 expression among PBMC cultured in the presence of 100 nM HMBPP and 100 U/ml IL-2 (gray) or absence of HMBPP and 10 U/ml IL-2 (black). Significant Vδ2 cell expansion was detected from day 5. Mean ± SEM of three different experiments is shown. (B) Sorting does not affect Vδ2 cell expansion. Parallel 6-d culture of bulk PBMC and sorted PBMC in the presence of HMBPP and IL-2 showing no significant differences of HMBPP-mediated Vδ2 cell expansion.

FIGURE 1.

Vδ2 cell expansion in response to HMBPP within bulk PBMC. (A) Kinetics of the percentage of Vδ2 expression among PBMC cultured in the presence of 100 nM HMBPP and 100 U/ml IL-2 (gray) or absence of HMBPP and 10 U/ml IL-2 (black). Significant Vδ2 cell expansion was detected from day 5. Mean ± SEM of three different experiments is shown. (B) Sorting does not affect Vδ2 cell expansion. Parallel 6-d culture of bulk PBMC and sorted PBMC in the presence of HMBPP and IL-2 showing no significant differences of HMBPP-mediated Vδ2 cell expansion.

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It was previously reported that most human cells are able to present some pyrophosphates and concluded that these cell types are able to expand γδT cells (10). Nevertheless, HMBPP has not been systematically tested using ex vivo purified primary Vδ2 cells, and, therefore, we wanted to identify the specific cellular requirements for HMBPP-mediated Vδ2 cell expansion. To precisely address the expansion of Vδ2 cells under different coculture conditions, all experiments started with a fixed number of sorted cell populations. First, we assessed whether cell populations expressing or not MHC-class II were comparable in their capacity to expand Vδ2 cells. After 6 d on culture, only in the cocultures with MHC-class II+ cells an expansion of Vδ2 cells could be measured (Fig. 2), indicating that MHC-class II+ cells are required to promote the expansion of Vδ2 cells mediated by HMBPP, whereas MHC-class II cells lack this capacity.

FIGURE 2.

MHC-class II+ cells are required for HMBPP-mediated Vδ2 cell expansion. A total of 20,000 purified Vδ2 cells was cocultured with either 80,000 purified MHC-class II+ or MHC-class II cells in the presence of HMBPP and IL-2. (A) Only MHC-class II+ cells promoted the expansion of Vδ2 cells, whereas MHC-class II were unable. Mean values of absolute Vδ2 cells of four different experiments are shown. Total Vδ2 cells present at the beginning of the culture are represented with black bars, and after 6-d culture with gray bars. (B) Representative dot plots of the percentage of Vδ2 cells after 6 d of coculture with MHC-class II+ cells (top) and MHC-class II cells. Percentage of Vδ2 cells in the beginning of the culture was 20%.

FIGURE 2.

MHC-class II+ cells are required for HMBPP-mediated Vδ2 cell expansion. A total of 20,000 purified Vδ2 cells was cocultured with either 80,000 purified MHC-class II+ or MHC-class II cells in the presence of HMBPP and IL-2. (A) Only MHC-class II+ cells promoted the expansion of Vδ2 cells, whereas MHC-class II were unable. Mean values of absolute Vδ2 cells of four different experiments are shown. Total Vδ2 cells present at the beginning of the culture are represented with black bars, and after 6-d culture with gray bars. (B) Representative dot plots of the percentage of Vδ2 cells after 6 d of coculture with MHC-class II+ cells (top) and MHC-class II cells. Percentage of Vδ2 cells in the beginning of the culture was 20%.

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Because the capacity of a specific population could be masked within the bulk MHC-class II cells, we next compared the ability of conventional αβTCR+ cells with MHC-class II+ cells to efficiently promote the expansion of Vδ2 cells. However, only MHC-class II+ cells were able to expand primary Vδ2 cells, whereas highly purified αβTCR+ cells failed completely (Fig. 3A, 3B). Furthermore, to ensure that the capacity of T cell subpopulations was not underestimated within bulk αβTCR+ cells, we performed cocultures of highly purified primary CD4+ or CD8+ T cells with Vδ2 cells. In addition, it was also reported that Vδ2 cell clones are able to present pyrophosphates to themselves (10), and, therefore, Vδ2 cells were cultured alone in the presence of HMBPP and IL-2. Cocultures of MHC-class II+ and MHC-class II cells were used as positive and negative controls, respectively. CD4+ and CD8+ T cells completely failed to promote Vδ2 cell HMBPP-mediated expansion, as well as cultures of highly purified Vδ2 cells alone. Significant expansion was only detected when MHC-class II+ cells were present (Fig. 3C).

FIGURE 3.

Inability of classical αβTCR+ cells and Vδ2 T cells to present HMBPP. (A) Purified conventional αβTCR+ T cells failed to promote Vδ2 cell expansion, whereas significant expansion was detected in the coculture with MHC-class II+ cells in response to HMBPP and IL-2. (B) Representative dot plots of the percentages of Vδ2 cells present in the cocultures with αβTCR+ cells and with MHC-class II+ cells. Percentage of Vδ2 cells in the beginning of the culture was 20%. (C) Sorted CD4+, CD8+, Vδ2 cells, and total MHC-class II cells lack the capacity to expand Vδ2 cells, whereas only MHC-class II+ cells induced their expansion. (D) Treg are not responsible for the inability of CD4 T cells to induce Vδ2 cell expansion. Vδ2 cells were cultured in the presence of sorted CD4+ cells depleted or not of the CD25+ fraction (containing Treg), and MHC-class II+ and MHC-class II cells as controls. In both cocultures with CD4+CD25+ and CD4+CD25 Vδ2 cells did not expand in the presence of HMBPP and IL-2. Total Vδ2 cells present at the beginning of the culture are represented with black bars, and after 6 d in culture with gray bars. Mean ± SEM of four different experiments is shown.

FIGURE 3.

Inability of classical αβTCR+ cells and Vδ2 T cells to present HMBPP. (A) Purified conventional αβTCR+ T cells failed to promote Vδ2 cell expansion, whereas significant expansion was detected in the coculture with MHC-class II+ cells in response to HMBPP and IL-2. (B) Representative dot plots of the percentages of Vδ2 cells present in the cocultures with αβTCR+ cells and with MHC-class II+ cells. Percentage of Vδ2 cells in the beginning of the culture was 20%. (C) Sorted CD4+, CD8+, Vδ2 cells, and total MHC-class II cells lack the capacity to expand Vδ2 cells, whereas only MHC-class II+ cells induced their expansion. (D) Treg are not responsible for the inability of CD4 T cells to induce Vδ2 cell expansion. Vδ2 cells were cultured in the presence of sorted CD4+ cells depleted or not of the CD25+ fraction (containing Treg), and MHC-class II+ and MHC-class II cells as controls. In both cocultures with CD4+CD25+ and CD4+CD25 Vδ2 cells did not expand in the presence of HMBPP and IL-2. Total Vδ2 cells present at the beginning of the culture are represented with black bars, and after 6 d in culture with gray bars. Mean ± SEM of four different experiments is shown.

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Because it was previously published that conventional CD4+CD25+ T regulatory cells (Treg) are able to inhibit γδ T cell proliferation (20), it was possible that their presence was responsible for the lack of Vδ2 cell expansion in cultures with purified CD4+ T cells. Therefore, purified CD4+CD25 (depleted of Treg) and CD4+CD25+ (enriched Treg) T cell subsets were cocultured with Vδ2 cells. The presence of CD4+CD25+ cells was not responsible for the lack of Vδ2 cell HMBPP-mediated expansion because CD4+CD25 T cells depleted of CD4+CD25+ T cells also failed to expand γδT cells (Fig. 3D).

Next, we questioned which MHC-class II+ subpopulation was most effective presenting HMBPP for Vδ2 cell expansion. For this set of experiments, the potential of sorted DC, B cells, and monocytes to promote a significant expansion of Vδ2 cells was compared. DC are most effective enhancing Vδ2 cell expansion when compared with B cells or monocytes (Fig. 4A, 4B). Interestingly, B cells showed only limited capacity to promote the expansion of Vδ2 cells. In addition, to assess that the high DC numbers present in the cocultures were not overestimating their ability to expand Vδ2 cells, cocultures were performed at 1:4, 1:2, and 1:1 ratios (Vδ2:other cell type). Results confirmed that DC were more potent expanders than monocytes or B cells (Fig. 4C). Furthermore, to independently confirm that DC exhibit the greatest capacity to restore Vδ2 cell expansion, MHC-class II+ depletion experiments were performed and subsets were added back. MHC-class II+ cells were depleted (Supplemental Fig. 1), and afterward, isolated autologous DC, monocytes, or B cells were added back to ensure that only one type of APC was present. In addition, PBMC depleted of MHC-class II+ cells (MHC-class II cells) were used as a negative control. Results of these experiments confirmed that DC promote stronger Vδ2 cell expansion than monocytes (p = 0.015) or B cells (p < 0.001) (Fig. 4D). Taken together, these data demonstrate that DC represented the most active MHC-class II+ subpopulation promoting primary Vδ2 cell expansion.

FIGURE 4.

DC are the most effective APC enhancing HMBPP-mediated Vδ2 cell expansion. (A) Comparison of the capacity of different MHC-class II+ subpopulations (DC, CD14+ monocytes, B cells) to promote Vδ2 cell expansion in response to HMBPP and IL-2. DC are superior to CD14+ cells (p < 0.001) promoting Vδ2 cell expansion, whereas B cells exhibited only limited capacity. Total Vδ2 cells present at the beginning of the culture are represented with black bars, and after 6 d in culture with gray bars. (B) Representative flow cytometry plots of the final Vδ2 cell percentage in the cocultures after 6 d in the presence of HMBPP and IL-2. (C) Titration of MHC-class II+ cell subset numbers. Cocultures of purified Vδ2 cells were performed at 1:4, 1:2, and 1:1 ratios. (D) Depletion experiments. PBMC were depleted of MHC-class II+ cells, and afterward, DC, CD14+ monocytes, or CD19+ B cells from the same donor were added back to the culture. DC capacity to restore Vδ2 cell expansion was greater than CD14+ and CD19+ cells, whereas MHC-class II cells were used as a negative control. Mean ± SEM of four (A) and three (C, D) different experiments is shown.

FIGURE 4.

DC are the most effective APC enhancing HMBPP-mediated Vδ2 cell expansion. (A) Comparison of the capacity of different MHC-class II+ subpopulations (DC, CD14+ monocytes, B cells) to promote Vδ2 cell expansion in response to HMBPP and IL-2. DC are superior to CD14+ cells (p < 0.001) promoting Vδ2 cell expansion, whereas B cells exhibited only limited capacity. Total Vδ2 cells present at the beginning of the culture are represented with black bars, and after 6 d in culture with gray bars. (B) Representative flow cytometry plots of the final Vδ2 cell percentage in the cocultures after 6 d in the presence of HMBPP and IL-2. (C) Titration of MHC-class II+ cell subset numbers. Cocultures of purified Vδ2 cells were performed at 1:4, 1:2, and 1:1 ratios. (D) Depletion experiments. PBMC were depleted of MHC-class II+ cells, and afterward, DC, CD14+ monocytes, or CD19+ B cells from the same donor were added back to the culture. DC capacity to restore Vδ2 cell expansion was greater than CD14+ and CD19+ cells, whereas MHC-class II cells were used as a negative control. Mean ± SEM of four (A) and three (C, D) different experiments is shown.

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To assess whether the capacity of MHC-class II+ cells to expand Vδ2 cells was restricted to the use of HMBPP or whether this effect could be generalized to other pyrophosphates, sorted Vδ2 cells were cocultured with MHC-class II+ or MHC-class II cells and incubated with either HMBPP or IPP for 6 d. Vδ2 cells expanded only in the presence of MHC-class II+ cells regardless of HMBPP or IPP presence (Fig. 5). These results indicated that MHC-class II+ cells are indispensable for HMBPP- or IPP-mediated Vδ2 cell expansion.

FIGURE 5.

IPP requires MHC-class II+ cells to expand Vδ2 cells. (A) MHC-class II cells are unable to promote IPP-mediated expansion of Vδ2 cells. Vδ2 cells were cocultured in the presence of IPP (1 μM) or HMBPP (100 nM) with highly purified MHC-class II+ or MHC-class II cells. Only MHC-class II+ cells have the capacity to promote the expansion of Vδ2 cells. Total Vδ2 cells present at the beginning of the culture are represented with black bars, and after 6 d in culture with gray bars. Mean ± SEM of four different experiments is shown. (B) Representative flow cytometry plots of the final Vδ2 cell percentages present in the culture after 6 d in the presence of IPP (starting Vδ2 cell frequency was 20%).

FIGURE 5.

IPP requires MHC-class II+ cells to expand Vδ2 cells. (A) MHC-class II cells are unable to promote IPP-mediated expansion of Vδ2 cells. Vδ2 cells were cocultured in the presence of IPP (1 μM) or HMBPP (100 nM) with highly purified MHC-class II+ or MHC-class II cells. Only MHC-class II+ cells have the capacity to promote the expansion of Vδ2 cells. Total Vδ2 cells present at the beginning of the culture are represented with black bars, and after 6 d in culture with gray bars. Mean ± SEM of four different experiments is shown. (B) Representative flow cytometry plots of the final Vδ2 cell percentages present in the culture after 6 d in the presence of IPP (starting Vδ2 cell frequency was 20%).

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The mechanism of HMBPP presentation to and recognition by Vδ2 cells is largely unknown. We further investigated whether HMBPP selectively interacts to a putative cell-associated molecule present on MHC-class II+ APC. Thus, MHC-class II+ and MHC-class II cells were incubated with HMBPP for 1 h either at 37°C or 4°C, extensively washed to avoid the presence of free HMBPP, and cocultured with the purified Vδ2 cells in the presence of IL-2. After 6 d on culture, Vδ2 cell expansion was detected only in the culture with MHC-class II+ cells, but not in the culture with MHC-class II cells, irrespective of the HMBPP-pulsing condition strategy (Fig. 6). In addition, to assess whether HMBPP needs to interact with intact cells, MHC-class II+ and MHC-class II cells were lysed after 1-h incubation with HMBPP and cocultured with Vδ2 cells in the presence of 100 U/ml IL-2. However, neither HMBPP-pulsed MHC-class II+ nor MHC-class II cell lysates were able to promote the expansion of Vδ2 cells (Fig. 6). Additionally, both MHC-class II + and MHC-class II cells were fixed with paraformaldehyde after HMBPP pulsing; however, after 6 d of culture, Vδ2 cell expansion was not detected (data not shown). These results indicate that Vδ2 cell expansion depends on HMBPP interaction with intact, nonfixed MHC-class II+ cells, and additionally, that viable HMBPP-pulsed MHC-class II+ cells can transfer Vδ2 expansion capacity in the absence of free HMBPP.

FIGURE 6.

HMBPP can be pulsed on MHC-class II+ cells and transforms MHC-class II+ into Vδ2 cell expanders. (A) MHC-class II+ and MHC-class II cells were pulsed with HMBPP for 1 h at 37°C or on ice, extensively washed, and cultured with purified Vδ2 cells in the presence of IL-2. Vδ2 cell expansion was comparable between MHC-class II+ cells pulsed at 37°C or on ice, as well as within the normal control (HMBPP and IL-2 added to the coculture together at basal time point). Lysis of HMBPP-pulsed MHC-class II+ cells abrogated their capacity to expand Vδ2 cells. HMBPP-pulsed MHC-class II cells were unable to expand Vδ2 cells in the presence of IL-2 irrespective of pulsing procedure. Total Vδ2 cells present at the beginning of the culture are represented with black bars, and after 6 d in culture with gray bars. Mean ± SEM of three different experiments is shown. (B) Examples of Vδ2 cell percentage of the flow cytometry analysis after 6 d of coculture with HMBPP-pulsed MHC-class II+ and MHC-class II cells either at 37°C or on ice (starting Vδ2 cell frequency was 20%).

FIGURE 6.

HMBPP can be pulsed on MHC-class II+ cells and transforms MHC-class II+ into Vδ2 cell expanders. (A) MHC-class II+ and MHC-class II cells were pulsed with HMBPP for 1 h at 37°C or on ice, extensively washed, and cultured with purified Vδ2 cells in the presence of IL-2. Vδ2 cell expansion was comparable between MHC-class II+ cells pulsed at 37°C or on ice, as well as within the normal control (HMBPP and IL-2 added to the coculture together at basal time point). Lysis of HMBPP-pulsed MHC-class II+ cells abrogated their capacity to expand Vδ2 cells. HMBPP-pulsed MHC-class II cells were unable to expand Vδ2 cells in the presence of IL-2 irrespective of pulsing procedure. Total Vδ2 cells present at the beginning of the culture are represented with black bars, and after 6 d in culture with gray bars. Mean ± SEM of three different experiments is shown. (B) Examples of Vδ2 cell percentage of the flow cytometry analysis after 6 d of coculture with HMBPP-pulsed MHC-class II+ and MHC-class II cells either at 37°C or on ice (starting Vδ2 cell frequency was 20%).

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We investigated the effect of blocking MHC-class II expression and the secondary induction of MHC-class II on CD4+ T cells to address the specific role of MHC-class II for Vδ2 cell expansion (Fig. 7). Blocking MHC-class II with a HLA-DR,DQ,DP mAb suppressed the capacity of purified primary MHC-class II+ cells to mediate Vδ2 cell expansion in comparison with isotype mAb control (Fig. 7A). Because these primary MHC-class II+ cells were FACS sorted with mAbs and the isotype control mAb also exhibited capacity to inhibit Vδ2 cell expansion, we performed additional experiments with untouched PBMC and further reduced the concentration of the blocking mAbs. These experiments confirmed that blocking primary MHC-class II+ cells significantly suppressed Vδ2 expansion in comparison with isotype controls (Fig. 7B). These experiments confirmed that MHC-class II molecules, directly or indirectly, may be involved in Vδ2 expansion. In contrast, secondary induction of MHC-class II on CD4+ T cells was unable to induce Vδ2 cell expansion, indicating that MHC-class II molecules alone are necessary, but not sufficient to promote Vδ2 cell expansion (Fig. 7C–E). To control for a general impairment of DC function in the presence of the blocking MHC-class II mAbs (1 μg/ml), we performed additional proliferation experiments with magnetic-bead–sorted naive CD8+ T cells cocultured with FACS-sorted allogeneic CD1c+ DC. These experiments indicated that DC-induced proliferation of naive CD8+ T cells was not impaired in the presence of the blocking MHC-class II mAb in comparison with isotype controls (Fig. 8).

FIGURE 7.

Primary MHC-class II+ cells are necessary for HMBPP-mediated Vδ2 cell expansion. (A) A total of 20,000 purified Vδ2 cells was cocultured with 80,000 purified MHC-class II+ cells in the presence of pan MHC-class II mAb, isotype control mAb, or without mAb (positive control) with HMBPP and IL-2. Total Vδ2 cells present at the beginning of the culture are represented with black bars, and after 6 d in culture with gray bars. (B) A total of 80,000 untouched PBMC was cultured in the presence of pan MHC-class II mAb, isotype control mAb, without mAb (positive control), or without HMBPP (negative control) with IL-2. (C and D) MHC-class II expression on unstimulated or CD3/CD28-stimulated purified CD4+ T cells. (E) A total of 20,000 purified Vδ2 cells was cocultured with 80,000 unstimulated CD4+ T cells (MHC-class II CD4+ T cells), CD3/CD28-stimulated CD4+ T cells (MHC-class II+ CD4+ T cells), and purified primary MHC-class II+ cells (positive control) in the presence of HMBPP and IL-2. Total Vδ2 cells present at the beginning of the culture are represented with black bars, and after 6 d in culture with gray bars. Only primary MHC-class II+ cells promoted the expansion of Vδ2 cells, whereas secondary induction of MHC-class II+ was unable to promote Vδ2 cell expansion capacity. Mean ± SEM of n = 8 (A, B), n = 3 (C), and n = 6 (E) different experiments is shown.

FIGURE 7.

Primary MHC-class II+ cells are necessary for HMBPP-mediated Vδ2 cell expansion. (A) A total of 20,000 purified Vδ2 cells was cocultured with 80,000 purified MHC-class II+ cells in the presence of pan MHC-class II mAb, isotype control mAb, or without mAb (positive control) with HMBPP and IL-2. Total Vδ2 cells present at the beginning of the culture are represented with black bars, and after 6 d in culture with gray bars. (B) A total of 80,000 untouched PBMC was cultured in the presence of pan MHC-class II mAb, isotype control mAb, without mAb (positive control), or without HMBPP (negative control) with IL-2. (C and D) MHC-class II expression on unstimulated or CD3/CD28-stimulated purified CD4+ T cells. (E) A total of 20,000 purified Vδ2 cells was cocultured with 80,000 unstimulated CD4+ T cells (MHC-class II CD4+ T cells), CD3/CD28-stimulated CD4+ T cells (MHC-class II+ CD4+ T cells), and purified primary MHC-class II+ cells (positive control) in the presence of HMBPP and IL-2. Total Vδ2 cells present at the beginning of the culture are represented with black bars, and after 6 d in culture with gray bars. Only primary MHC-class II+ cells promoted the expansion of Vδ2 cells, whereas secondary induction of MHC-class II+ was unable to promote Vδ2 cell expansion capacity. Mean ± SEM of n = 8 (A, B), n = 3 (C), and n = 6 (E) different experiments is shown.

Close modal
FIGURE 8.

MHC-class II mAb does not induce a general impairment of DC function. (A and B) A total of 100,000 magnetic-bead–purified, CFSE-labeled naive CD8+ T cells was cocultured with FACS-sorted allogeneic CD1c+ DC at the indicated ratios in the presence of pan MHC-class II mAb (1 μg/ml) for 5 d. Controls included CD1c+ DC cultured in parallel from the same donor in the presence of isotype control mAb (1 μg/ml). Naive CD8+ T cell proliferation controls included CD3+CD28+-stimulated cells (positive control) and naive CD8+ T cells cultured alone without DC (negative control) to control for unspecific T cell proliferation. Mean ± SEM of n = 3 different experiments is shown.

FIGURE 8.

MHC-class II mAb does not induce a general impairment of DC function. (A and B) A total of 100,000 magnetic-bead–purified, CFSE-labeled naive CD8+ T cells was cocultured with FACS-sorted allogeneic CD1c+ DC at the indicated ratios in the presence of pan MHC-class II mAb (1 μg/ml) for 5 d. Controls included CD1c+ DC cultured in parallel from the same donor in the presence of isotype control mAb (1 μg/ml). Naive CD8+ T cell proliferation controls included CD3+CD28+-stimulated cells (positive control) and naive CD8+ T cells cultured alone without DC (negative control) to control for unspecific T cell proliferation. Mean ± SEM of n = 3 different experiments is shown.

Close modal

To further characterize constitutive MHC-class II involvement on Vδ2 cell expansion, we analyzed the capacity of different cell lines to mediate expansion (Fig. 9). Daudi and Raji cells expressed high levels of MHC-class II (>90%), whereas THP-1 cells (≤10%) and U937 cells (<2%) expressed very low or no detectable MHC-class II (Fig. 9A–C). Additionally, the MHC-class II–negative, non-APC cell lines KG-1a and MEG-01 were analyzed. Results indicated that only the MHC-class II+ Daudi cells expanded Vδ2 cells, whereas the other cell lines, including Raji cells, failed to expand Vδ2 cells (Fig. 9D). These results again suggested that MHC-class II expression is necessary, but not sufficient to promote Vδ2 cell expansion.

FIGURE 9.

Heterogenous capacity of MHC-class II–positive and –negative cell lines to mediate Vδ2 cell expansion. (A) Representative dot plots showing MHC-class II expression (HLA-DR, DP, DQ) in comparison with isotype control mAb on MHC-class II+ (Daudi) and MHC-class II (KG-1a) cell lines. (B and C) MHC-class II HLA-DR and HLA-DR, DQ, DP surface expression on cell lines. (D) Mean absolute Vδ2 cells after coculture of 20,000 purified Vδ2 cells with 80,000 purified cell line cells in the presence of HMBPP and IL-2. Total Vδ2 cells present at the beginning of the culture are represented with black bars, and after 6 d in culture with gray bars. Only Daudi cells promoted the expansion of Vδ2 cells. Mean ± SEM of n = 3 (B, C) and n ≥ 7 (D) different experiments is shown.

FIGURE 9.

Heterogenous capacity of MHC-class II–positive and –negative cell lines to mediate Vδ2 cell expansion. (A) Representative dot plots showing MHC-class II expression (HLA-DR, DP, DQ) in comparison with isotype control mAb on MHC-class II+ (Daudi) and MHC-class II (KG-1a) cell lines. (B and C) MHC-class II HLA-DR and HLA-DR, DQ, DP surface expression on cell lines. (D) Mean absolute Vδ2 cells after coculture of 20,000 purified Vδ2 cells with 80,000 purified cell line cells in the presence of HMBPP and IL-2. Total Vδ2 cells present at the beginning of the culture are represented with black bars, and after 6 d in culture with gray bars. Only Daudi cells promoted the expansion of Vδ2 cells. Mean ± SEM of n = 3 (B, C) and n ≥ 7 (D) different experiments is shown.

Close modal

To assess whether certain cytokines were preferentially produced after Vδ2 cell expansion, a set of different cytokines was measured in the supernatants of cocultures of Vδ2 cells either with MHC-class II+ or MHC-class II cells. Anti-inflammatory cytokines IL-4 and IL-10; proinflammatory cytokines IL-6, IFN-γ, TNF-α, and IL-17; and cytokines involved in cell proliferation IL-9 and IL-15 were significantly elevated when Vδ2 cells expanded in the presence of MHC-class II+ cells compared with cocultures with MHC-class II cells (p < 0.001 in all cases), although at different rates (Fig. 10). IL-4 and IL-10 production was detected after Vδ2 cell expansion. Interestingly, IL-6 exhibited extremely elevated production. As expected, IFN-γ and TNF-α were produced only when Vδ2 cells were expanded.

FIGURE 10.

Association of HMBPP-mediated Vδ2 cell expansion with cytokine production. Cytokines were measured in the supernatants of Vδ2 cells cultured with MHC-class II+ or MHC-class II cells after 6 d. All cytokines showed a higher production (pg/ml) in cultures and were Vδ2 cells expanded in the presence of MHC-class II+ cells when compared with cocultures with MHC-class II cells (p < 0.001 in all cases). Cultures and cytokine measurement were performed in duplicate. Mean ± SEM of four different experiments is shown.

FIGURE 10.

Association of HMBPP-mediated Vδ2 cell expansion with cytokine production. Cytokines were measured in the supernatants of Vδ2 cells cultured with MHC-class II+ or MHC-class II cells after 6 d. All cytokines showed a higher production (pg/ml) in cultures and were Vδ2 cells expanded in the presence of MHC-class II+ cells when compared with cocultures with MHC-class II cells (p < 0.001 in all cases). Cultures and cytokine measurement were performed in duplicate. Mean ± SEM of four different experiments is shown.

Close modal

Mechanisms of P-Ag presentation to and recognition by Vδ2 cells remain poorly understood. We systematically assessed cellular requirements of Vδ2 cell HMBPP-mediated expansion using highly purified primary cells. We demonstrate that primary MHC-class II+ cells are necessary for HMBPP-driven human Vδ2 cell expansion, whereas, surprisingly, MHC-class II cells are unable to mediate their expansion. This lack of MHC-class II cells’ capacity to expand Vδ2 cells was also confirmed for IPP. In addition, among MHC-class II+ APC, DC have a superior capacity compared with monocytes or B cells promoting Vδ2 cell expansion. Finally, we demonstrate that HMBPP selectively interacts with a putative molecule present in MHC-class II+ cells that might be absent in MHC-class II cells. Moreover, our experiments indicate that HMBPP needs to interact with viable MHC-class II+ cells to promote Vδ2 expansion. MHC-class II–blocking experiments with mAb impaired Vδ2 cell expansion, whereas secondary MHC-class II induction on CD4+ T cells was not able to revert the inability of T cells to promote Vδ2 cell expansion. Accordingly, it should be noted that these results do not provide a formal proof regarding the molecular significance of MHC-class II, but rather suggest that MHC-class II expression is necessary, but not sufficient for Vδ2 cell expansion.

Previous studies about the cellular requirements of Vδ2 cells to expand in response to pyrophosphates reported that nearly each cell type, even CD4+ and CD8+ T cells, is able to induce Vδ2 cell proliferation (10). Nevertheless, in this previous study, cell lines and γδT cell clones were used and might explain, at least partially, the differences with our results. In addition, the sorting procedure did not modify either Vδ2 cells’ capacity to expand or APC capacity to present the HMBPP, regardless of the procedural stress. Our results demonstrate that the mechanism of pyrophosphate, that is, HMBPP and IPP, presentation requires MHC-class II+ cells to promote the expansion of Vδ2 cells. MHC-class II cells, including CD4+ and CD8+ T cells, lack this capacity, and even purified primary Vδ2 cells were not able to present HMBPP to themselves. We further assessed whether Treg were responsible for the inability of CD4+ T cells to expand Vδ2 cells, because it was previously published that Treg can inhibit Vδ2 cell IFN-γ production (21) and their proliferation (20) mediated by bromohydrin pyrophosphate. Results indicate that the presence of CD4+CD25+ Treg was not responsible for inability of CD4+ T cells to expand Vδ2 cells in response to HMBPP.

A previous report suggested that HMBPP could be presented by macaque B cells and CD8+ T cells using a Vγ9Vδ2 tetramer construct (12). In this study, we further demonstrate that human B cells have a limited capacity to promote an HMBPP-mediated Vδ2 cell expansion, whereas human CD8+ T cells failed completely. According to our results, although using bisphosphonates, it was reported that Vδ2 cells were activated and produced IFN-γ only in the presence of adherent cells of monocyte lineage (15). Among the MHC-class II+ APC, DC are more potent than monocytes or B cells promoting HMBPP-mediated Vδ2 cell expansion. DC are capable of enhancing in vitro activation and proliferation of other innate-like cells (22, 23). In this regard, several studies reported functional interactions between P-Ag or bisphosphonate-activated Vδ2 cells and DC (16, 2429). Moreover, Vδ2 cell function is impaired after Mycobacterium tuberculosis infection of DC (30). In previous reports, TLR-activated DC were found to play a critical role promoting high IFN-γ production in Vδ2 cells (31). Our results further underline the importance of DC-Vδ2 crosstalk by showing that DC represent the most effective cell type promoting P-Ag–mediated Vδ2 cell expansion. Accordingly, subsets of DC are required for PAM (25) and zoledronate-mediated (27) γδT cell expansion. Interestingly, by performing back-adding experiments, we found that DC completely restored the inability of MHC-class II+–depleted PBMC to expand Vδ2 cells.

In previous reports, IPP could not be pulsed on EBV-transformed B cells to promote the expansion of different Vδ2 cell clones (10). This could be partially explained because we demonstrate in this work the restricted capacity of B cells to promote the expansion of Vδ2 cells. However, later reported, synthetic pyrophosphates, as well as PAM and Zol (11, 14, 27, 32, 33), were pulsed on tumor cell lines that activated Vδ2 cells. In addition, using a Vγ9Vδ2 tetramer construct, it was suggested that HMBPP could be pulsed on APC (12). By using sorted primary cells, we have shown in this study that HMBPP can efficiently be pulsed only on viable MHC-class II+ cells. Therefore, it is possible that HMBPP stably associates with a putative molecule present in the surface of MHC-class II+ cells, but absent on MHC-class II cells. There is accumulating evidence that P-Ag are displayed on cellular membranes for T cell recognition (11, 12). This is supported by our results because MHC-class II+ cell capacity to promote primary Vδ2 cell expansion was abrogated after either fixation with paraformaldehyde or lysis by freeze-thaw cycles. Therefore, primary viable MHC-class II+ cells are required for efficient HMBPP-mediated expansion of Vδ2 cells.

In addition to our primary objective, we have also analyzed the role of MHC-class II molecule on Vδ2 cell expansion by using cell lines with different levels of MHC-class II expression, by blocking its expression on primary sorted cells and bulk PBMC, and also by inducing secondary MHC-class II expression on CD4 T cells. Our results demonstrate that other putative molecule present in primary MHC-class II+ cells might be required to promote the expansion of Vδ2 cells, whereas mere expression of MHC-class II is not sufficient. Further studies are required to establish the mechanisms of P-Ag presentation by MHC-class II–expressing cells to Vδ2 cells.

Specific cytokines exert striking and contrasting immunologic effects in response to pathogens, and, thus, it is important to characterize the cytokines associated with efficient Vδ2 cell expansion in response to HMBPP. It was previously reported that HMBPP and IL-2 treatment during simian-HIV infection in vivo was associated with the increased production of IFN-γ, TNF-α, IL-4, and IL-10 (18, 34). In a healthy ex vivo context, we confirm an increased production of these cytokines after Vδ2 cell expansion in response to HMBPP and further demonstrate the production of IL-6, IL-9, IL-15, and IL-17. Interestingly, the proinflammatory cytokine IL-6 was secreted at 100- to 1000-fold elevated levels in cultures with expanding Vδ2 cells. Monocytes/Macrophages are the main source of IL-6 (35), and, therefore, it would be interesting to study further the reciprocal relationship between them and γδT cells after treatment with HMBPP because it was reported that Plasmodium falciparum infection induced the production of IL-6 and TNF-α by γδT cells (36). In addition, Th17 cytokines are strongly implicated in host immunity to both extracellular and certain intracellular pathogens. IL-17, through its effects on DC, is important for Th1 immunity against intracellular pathogens M. tuberculosis and Francisella tulerensis (37, 38). In many infections, γδ T cell responses are the predominant source of IL-17 (39, 40), and, in line with our results, IL-17–producing γδ T cells selectively expanded in response to pathogen products (39). Finally, we report in this work the production of IL-9 and IL-15 in cultures in which Vδ2 cells expanded in the presence of MHC-class II+ cells. Among others, IL-9 and IL-15 have a direct effect on the regulation of γδ T cell homeostasis and IL-9 is involved in pathogen clearance (41). In summary, we report the production of several cytokines after Vδ2 cell activation and expansion, specifically in the presence of MHC-class II+ cells. However, the functional role of these cytokines on Vδ2 cell and MHC-class II+ cells deserves further investigation.

Vδ2 cells have arisen as promising targets for different immunotherapies (4245). Despite recent progress understanding the mechanism of P-Ag presentation to γδ T cells, little is known with respect to the primary cellular requirements to promote their expansion. It is important to note that in the current study we have focused on primary resting Vδ2 cells (i.e., not pre-expanded primary γδ T cells) that may require both Ag presentation and costimulation, whereas for preactivated γδ T cells mere presentation of Ags may be sufficient for activation. Our results provide novel information about the specific cellular requirements for resting Vδ2 cell expansion mediated by HMBPP, and may be useful for defining future cellular therapy strategies.

We thank Gaby Haley, Angelika Nockher, and Nelli Baal for technical assistance.

This work was supported by the Excellence Cluster Cardio-Pulmonary System (to N.S.-S., T.K., and H.H.) and by the University of Gießen and Marburg Lung Center.

The online version of this article contains supplemental material.

Abbreviations used in this article:

DC

dendritic cell

HMBPP

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

IPP

isopentenyl pyrophosphate

P-Ag

phosphoantigen

PAM

pamidronate

Treg

T regulatory cell.

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The authors have no financial conflicts of interest.