Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized whole blood of healthy volunteer donors (red-cross Austria) by standard density-gradient centrifugation with Lymphoprep (07851, Axis-Shield PoC AS). Donors gave their written informed consent, and approval was obtained from the ethics committee of the Medical University of Vienna (ECS1183/2016). Monocytes were purified using MagniSort CD14 Separation Kits (8802-6834-74, eBioscience). Bulk T cells were purified using MACS Pan T Cell Isolation Kits (130-096-535, Miltenyi). Populations showed at least 95% purity. Cells were either immediately processed or cryopreserved in RPMI medium containing 10% FBS and 10% DMSO for later use. For the generation of immature and mature DCs, monocytes were cocultured with IL-4 (0.1 U/μl) and GM-CSF (50 ng/ml) for 5–6 days, as described previously (28). Mature DCs were generated by the addition of LPS (0.3 μg/ml) as a maturation stimulus for an additional 24 h. Melanoma patient samples were obtained from melanoma patients in regular care at the dermato-oncology out-patient clinic of the medical university of Vienna. The study was approved by the local ethics committee (1210/2012), and informed consent was obtained from the patients.

For T cell proliferation assays, 1–2 × 10⁷ T cells were labeled with 1 μl of a 1 mM CFSE stock solution (C34554, Molecular Probes) in 1 ml PBS for 4 min at room temperature. Subsequently, cells were washed twice with RPMI containing 10% FBS.

CFSE-labeled T cells (1 × 10⁵/well; 1 × 10⁶/ml) were then cocultured with 1.5 × 10³ or 6 × 10³/well monocyte-derived allogeneic DCs in 96-well round-bottom plates for 6 days, unless indicated otherwise. RPMI-1640 (R8758, Gibco) supplemented with 10% FBS, Penstrep, and Amphotericin was used as a standard cell culture medium.

The following monoclonal-blocking antibodies or combinations thereof were added at a final concentration of 10 μg/ml: TIGIT (clone MBSA43, eBioscience), CTLA-4 (Ipilimumab, Yervoy^®), Rat IgG_2A isotype, and TIM-3 (clones 54447 and 344823 from R&D Systems). Ligand-blocking antibodies to PD-1 (clone MK-3475), BTLA (clone E4H9), CD160 (clone CL1-R2), LAG-3 (clone 25F7), and an isotype control antibody [all human IgG1 carrying two mutations, L234A and L235A, in the CH2 domain of the heavy chain eliminating ADCC and CDC effector functions, as described elsewhere (29)] were produced from publicly available sequences by transient expression in CHO cells and purified using Mabselect SuRe-based affinity chromatography (GE Healthcare). The TIGIT mAb clone MBSA43 was shown previously to enhance HIV-1-specific T cell responses by blocking TIGIT inhibition (30). The TIM-3 antibody clone 344823 was previously used to block TIM-3 (31). The CD160 antibody CL1-R2 was described previously to block interaction with HVEM and enhance HIV-1-specific CD8 T cell responses (32, 33). BTLA Ab E4H9 and LAG-3 Ab 25F7 were validated in Jurkat-based reporter assays, as described in Figure S1 in Supplementary Material.

Culture supernatants were harvested after 6 days of coculture and stored at −20°C, followed by Luminex multiplex cytokine analysis (System 100, Luminex Inc.). The concentration of IFN-γ, IL-2, IL-10, IL-13, IL-17, and TNF-α was measured according to the manufacturer’s instructions.

Multicolor flow cytometry analysis was performed to assess proliferation and surface-marker expression of resting and activated T cells and DCs using the following mAbs: CD1a-PE (HI149), CD3-PE-Cy7 and BV421 (UCHT1), CD4-PerCP-Cy5.5 (RPA-T4), CD8-BV421 (RPA-T8), CD28-PE (CD28.2), CD66a-PE (ASL-32), CD80-PE (2D10), CD83-PE (HB15e), CD155-PE (SKII.4), CD160-PE and -APC (BY55), CD270-PE (122), CD272-APC (MIH26), CD273-PE (24F.10C12), CD274-PE (29E.2A3), CD279-APC (EH12.2H7), mouse IgG₁ isotype control-PE and -APC (MOPC-21), and mouse CD45.2-PE-Cy7 (104), all from Biolegend. CD1a-APC and -BV421 (HI149), CD4-PE (RPA-T4), CD8-APC and PE-Cy7 (RPA-T8), CD14-APC (M5E2), and CD25-PECy7 (M-A251) were purchased from BD Pharmingen. CD86-PE (IT2.2), CD152-eF660 (14D3), CD223-PE (3DS223H), CD258-PE (7-3), and TIGIT-PE (MBSA43) antibodies were from eBioscience, and CD366-PE (344823) from R&D Systems. Cells were stained in ice cold FACS buffer (PBS, 1% BSA, and 0.1% NaN₃) for 30 min. DCs were additionally incubated in 1 mg/ml Beriglobin (300666, CSL Behring) for 20 min on ice before staining to prevent non-specific binding of the mAbs to Fc receptors. 7-AAD (Biolegend) was used to exclude dead cells from the analysis.

Data were acquired on an LSR Fortessa (BD Biosciences) and analyzed using FlowJo 10.2 (Tree Star, Ashland, OR, USA). Fluorescence intensity is shown on a standard logarithmic or biexponential scale.

Effector T cells from allo-MLRs were harvested on day 6, counted, and incubated with target cells at a ratio of 5:1 or 1:1 for 2–3 h at 37°C before analysis by flow cytometry. Stimulator cells (mouse Bw5147 cells expressing membrane-bound aCD3, abbreviated Bw_aCD3) were used as target cells, as described previously (34). To determine the specific cell lysis, an equal number of wild-type Bw (Bw_wt) cells, which lack the expression of aCD3, was added. Bw_wt and Bw_aCD3 were discriminated by staining for CD14, contained in the stem of the aCD3 construct. Percentage of specific lysis was calculated using the following formula: 100−number of live BWaCD3*100number of live BWwt.

Data were normalized to an arbitrary scale using an adaptation of the standard score, calculated with the formula xnorm=x−baselineSD, where baseline means the median of the isotype control samples, and SD stands for the standard deviation of all the antibody conditions of a T cell donor. The normalization score shows the number of SDs by which the data point differs from the respective isotype control replicates.

All statistical analyses were performed using Graphpad Prism 7. Proliferation and cytokine data were analyzed with non-parametric repeated measurement ANOVAs (Friedman’s test). Dunn’s multiple comparison post hoc test was used to determine differences to the IgG1 isotype or PD-1 control condition. P values under 0.05 were considered significant (*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****).

We used cocultures of human CFSE-labeled T cells with allogeneic DCs to evaluate the capacity of immune checkpoint inhibitors targeting TIM-3, BTLA, CD160, TIGIT, LAG-3, and CTLA-4 alone or in combination with a PD-1 blocker to enhance human T cell responses. We selected antibody clones that were previously described to potently block interaction with their ligands, and in addition validated the BTLA and LAG-3 antibodies using a Jurkat-based reporter platform (as described in Section “Materials and Methods” and Figure S1 in Supplementary Material). Stimulation of T cells derived from a representative donor with (6 × 10³) mature DCs resulted in high percentages of CFSE^low T cells in the CD4 and CD8 subsets. Addition of a PD-1-blocking antibody enhanced T cell proliferation in both subsets, and a combination of immune checkpoint inhibitors targeting PD-1 and BTLA further augmented CFSE^low CD4 and CD8 T cells (Figure 1A). Analysis of data from 26 healthy donors underlines the potency of PD-1 to enhance CD4 and CD8 T cell proliferation (Figure 1B). Although a tendency of TIM-3 and BTLA antibodies to increase CD4 T cell proliferation was observed, blocking antibodies targeting co-inhibitors other than PD-1 generally failed to significantly increase T cell proliferation when used alone. By contrast, antibodies to several immune checkpoints had the capacity to further increase T cell proliferation when combined with PD-1 blockade. Antibodies to TIM-3 and LAG-3 augmented CD4 T cell proliferation, and the CTLA-4 antibody ipilimumab increased the proliferation in the CD8 subset when used in combination with PD-1 blockade. Importantly, the BTLA antibody was the only agent able to significantly enhance PD-1 effects in both CD4 and CD8 T cells. The addition of a blocking antibody to CD160, which shares the ligand HVEM with BTLA, did not further enhance the effect of BTLA blockade (Figure 1B). Although T cell proliferation in response to allogeneic stimulation depended on the number and maturation status of DCs, comparable effects of immune checkpoint inhibitors were also observed in cocultures with immature DCs or lower numbers of DCs (Figure S2 in Supplementary Material). Importantly, when using PBMC from patients with melanoma in allogeneic coculture assays, we could observe a similar tendency of the PD-1/TIM-3 and PD-1/BTLA combinations to enhance proliferation, with TIM-3 being particularly effective in both CD4 and CD8 T cells (Figure S3 in Supplementary Material).

In order to locate possible skews in the cytokine pattern, we analyzed the concentration of multiple cytokines in the culture supernatants of T cell proliferation assays using Luminex™-based multiplexing. Our results show that PD-1 blockade greatly increases the IFN-γ content in the culture supernatant (Figure 2A). Analysis of IL-2, IL-10, IL-13, IL-17a, and TNF-α pointed to a unique potency of PD-1 blockade to enhance cytokine production in T cell stimulation cultures (Figures 2B,C; Figure S4 in Supplementary Material). PD-1 in combination with TIGIT blockade reduced IL-2 and IL-13 levels compared to PD-1 blockade alone (Figure S4 in Supplementary Material). Blocking BTLA alone or together with PD-1 had the tendency to increase cytokine production but the effect did not reach statistical significance (Figure 2; Figure S4 in Supplementary Material).

It is evident that the efficacy of blocking antibodies critically depends on the expression of their antigens on T cells. Since it is known that immune checkpoints like PD-1, TIGIT, TIM-3, LAG-3, and also PD-L1 are strongly upregulated during T cell activation, we set out to monitor the expression of these markers in T cells stimulated with allogeneic DCs. As expected we observed a strong increase of CFSE^low and CD25⁺ T cells in the CD4 and CD8 subsets over the 6-day time course of the experiment (Figures 3A,B). PD-1 expression was absent in unstimulated CD4 T cells and low in unstimulated CD8 T cells. T cells strongly upregulated PD-1 when cultured in presence of allogeneic DCs, but not when cultured alone (Figure 3C). TIGIT expression on CD4 and CD8 T cells also increased with the duration of stimulation, but was not influenced by PD-1 blockade (Figure 3D). TIM-3, LAG-3, and PD-L1 were upregulated in both CD4 and CD8 T cells upon stimulation. Importantly, PD-1 blockade further increased the percentage of T cells positive for these molecules (Figures 3E–G). Furthermore, we found that PD-1, TIGIT, TIM-3, LAG-3, and PD-L1 expression was selectively upregulated in T cells that responded to allogeneic DCs, since these markers were mainly confined to T cells that had divided and expressed the activation marker CD25 (Figures 3E–G; data not shown). In contrast to the other immune checkpoints analyzed in our study, we observed that BTLA was expressed in the majority of freshly isolated T cells, which is in line with previous analyses (35). We observed a trend of BTLA downregulation in T cell cocultures with allogeneic DCs (data not shown).

The ultimate goal of immune checkpoint inhibitor-based therapies is to promote the eradication of tumors by effector cells. We chose to analyze PD-1 antibodies and immune checkpoint combinations that showed efficacy in enhancing CD8 T cell proliferation regarding their ability to promote cytotoxic effector function. T cells stimulated by allogeneic DCs for 6 days were harvested, counted, and cocultured with murine Bw5147 cells expressing a membrane-bound anti-CD3 antibody, which served as target cells for TCR complex-mediated killing as described before (34). Bw5147 cells lacking anti-CD3 expression were used to control for unspecific cytotoxic activity (Figure 4A). While T cells stimulated by allogeneic DCs in presence of PD-1 antibodies showed a trend to increase the percentage of specific killing, only the combined use of PD-1 and BTLA antibodies resulted in significantly increased killing compared to allo-stimulated T cells without the addition of a blocking antibody (Figures 4B,C).

It is well established that the maturation status of DCs has critical impact on their T cell stimulatory capacity. Upon LPS treatment, immature DCs strongly upregulated the maturation marker CD83 and also costimulatory ligands and MHC expression, which are crucial for their ability to induce vigorous T cell responses (Figure S5 in Supplementary Material; data not shown). Compared to immature DCs, the LPS-treated DCs induced increased proliferative responses in CD4 and in CD8 T cells (Figure 5A). We observed that immature DCs strongly upregulated the costimulatory molecules CD80, CD83, and CD86 and the coinhibitory markers CD155, PD-L1, and PD-L2 within 24–48 h of coculture (Figure 5B). As shown in Figure 5C, mature DCs also changed their expression profile during coculture, further upregulating PD-L1 and PD-L2. Interestingly, the presence of a PD-1-blocking antibody seemed to accelerate maturation, i.e., CD80 and CD83 expression, of iDCs. Furthermore, CD155 and PD-L1 expression was increased in presence of PD-1-blocking antibodies (Figure 5C).

Donor compatibility of T cells and allogeneic DCs, as well as coinhibitory ligand availability on DCs, might influence T cell proliferation in response to checkpoint inhibition. Since the effects of immune checkpoint inhibitors differed between cocultures, we performed a set of experiments to address whether these differential effects can be attributed to the DCs or the T cells in the cocultures. T cells derived from four different donors were each stimulated with DCs derived from three unrelated donors. The results were displayed by combining data from four different T cell donors stimulated with DCs from one individual donor (Figure 6A) and by combining data of individual T cells donors stimulated by DCs derived from three different donors (Figure 6B). The results indicate that T cells derived from different donors varied considerably regarding their response to different checkpoint inhibitors, whereas the choice of DC donor appeared to have minor impact.

In summary, we could show that single checkpoint inhibitors, except PD-1, do not greatly increase proliferation or change the cytokine pattern in healthy donors. In combination with PD-1, however, multiple checkpoint inhibitors were able to augment CD4 and CD8 T cell proliferation compared to PD-1 alone. We found that many of these checkpoint molecules are upregulated on T cells upon PD-1 blockade, and coinhibitory as well as costimulatory molecules are also enhanced on allo-DCs at the beginning of coculture and in response to PD-1 blockade.