Purpose: PD-1 checkpoint blockade has revolutionized the field of cancer immunotherapy, yet the frequency of responding patients is limited by inadequate T-cell priming secondary to a paucity of activatory dendritic cells (DC). DC signals can be bypassed by CD27 agonists, and we therefore investigated if the effectiveness of anti–PD-1/L1 could be improved by combining with agonist anti-CD27 monoclonal antibodies (mAb).

Experimental Design: The efficacy of PD-1/L1 blockade or agonist anti-CD27 mAb was compared with a dual-therapy approach in multiple tumor models. Global transcriptional profiling and flow cytometry analysis were used to delineate mechanisms underpinning the observed synergy.

Results: PD-1/PD-L1 blockade and agonist anti-CD27 mAb synergize for increased CD8+ T-cell expansion and effector function, exemplified by enhanced IFNγ, TNFα, granzyme B, and T-bet. Transcriptome analysis of CD8+ T cells revealed that combination therapy triggered a convergent program largely driven by IL2 and Myc. However, division of labor was also apparent such that anti–PD-1/L1 activates a cytotoxicity–gene expression program whereas anti-CD27 preferentially augments proliferation. In tumor models, either dependent on endogenous CD8+ T cells or adoptive transfer of transgenic T cells, anti-CD27 mAb synergized with PD-1/L1 blockade for antitumor immunity. Finally, we show that a clinically relevant anti-human CD27 mAb, varlilumab, similarly synergizes with PD-L1 blockade for protection against lymphoma in human–CD27 transgenic mice.

Conclusions: Our findings suggest that suboptimal T-cell invigoration in cancer patients undergoing treatment with PD-1 checkpoint blockers will be improved by dual PD-1 blockade and CD27 agonism and provide mechanistic insight into how these approaches cooperate for CD8+ T-cell activation. Clin Cancer Res; 24(10); 2383–94. ©2018 AACR.

This article is featured in Highlights of This Issue, p. 2235

Translational Relevance

Antitumor effector CD8+ T cells are often suppressed by PD-1 signaling. Although blocking antibodies to PD-1 and PD-L1 have shown impressive results in multiple tumor types, only a minority of patients respond, and recent findings suggest that stimulatory signals for T-cell activation may be lacking in some patients. We here show that agonist antibodies targeting mouse or human CD27, a key costimulatory receptor, act synergistically with anti–PD-1/L1 to increase CD8+ T-cell proliferation, effector function, and tumor clearance in wild-type and human–CD27 transgenic mice. Detailed mechanistic analysis indicates cooperative, yet also independent, influences of these antibodies on aspects of T-cell activation. Thus, our data reveal synergy between anti–PD-1/L1 and anti-CD27 for immunotherapy and provide renewed impetus for clinical evaluation of combined anti-CD27 and anti–PD-1/L1.

While it is clear that activated CD8+ T cells can be harnessed to kill tumor cells, T-cell activation is a complex, multifaceted process, tightly controlled by activatory and inhibitory receptors. To effectively utilize T cells for immunotherapy, it is therefore necessary to understand how these signals can be exploited to support appropriate T-cell activation. PD-1 is a well-studied inhibitory receptor triggered by binding to PD-L1 or PD-L2, and which counters T-cell activation through a mechanism dependent on recruitment of the protein tyrosine phosphatase SHP-2 to an immunoreceptor-based tyrosine switch motif (ITSM) in its cytoplasmic tail. Subsequent signaling events act to repress TCR-driven signals, inhibit the PI3K, Ras, and ERK signaling pathways, limit T-cell/dendritic cell (DC)-dwell time, and promote expression of the inhibitory transcription factor BATF (1). More recently, Hui and colleagues (2) showed that CD28 is the primary target of PD-1–recruited SHP-2, suggesting that PD-1 inhibits T-cell function through inactivation of CD28 signaling. The transient upregulation of PD-1 during T-cell activation and its maintenance at high levels on chronically stimulated (exhausted) T cells enable PD-1 to negatively regulate T-cell function during priming, secondary activation, and in conditions of prolonged antigen exposure (1, 3, 4). Furthermore, PD-1 is expressed on a large proportion of tumor-infiltrating T cells, yet even within this population, PD-1 expression is skewed toward the tumor-reactive, and presumably chronically stimulated, subpopulation (1, 5).

Encouragingly, countering PD-1 with PD-1– or PD-L1–specific monoclonal antibodies (mAb) enables functional CD8+ T cells to accumulate, leading to tumor rejection in preclinical models and dramatically improved outcomes across a range of cancer types in patients. Despite these unprecedented clinical successes, response rates remain modest and rarely exceed 40% (6). Reasons for the observed patient variability to PD-1/L1 blockade are doubtless multifactorial, but the extent of CD8+ T-cell infiltration is likely to be an important factor in the resistance to immunotherapy (7). Recent findings demonstrate that the lack of T-cell infiltration is the result of defective recruitment and activation of DCs, which leads to reduced cross-priming of CD8+ T cells (8). Based on these findings, a number of studies have shown that the intratumoral administration of inflammatory mediators alone or together with DCs can result in T-cell priming and improved antitumor immunity (8). However, the broad applicability of this approach, particularly for the treatment of metastatic disease, is likely to be limited. We and others have previously demonstrated that DC expression of CD70, the ligand for CD27, is required for efficient priming of CD8+ T cells and effective antitumor immunity in murine models (9–14). Furthermore, we have shown that the requirement for DC activation can be largely replaced by systemic administration of soluble recombinant CD70 or agonist anti-CD27 mAb (12, 14, 15). We reasoned that the ability of CD27 agonists to enhance CD8+ T-cell priming could combine with PD-1 blockade to further improve CD8+ T-cell responses and antitumor immunity. Indeed we have shown previously in a CD4-independent model of donor lymphocyte infusion in which CD8+ T cells are exhausted by continued exposure to antigen, that transient CD8+ T-cell reinvigoration can be achieved by the combination of agonist anti-CD27 and anti–PD-L1 (16). In contrast however, others have reported that agonist anti-CD27 and PD-1 blockade fails to synergize for improved CD8+ T-cell accumulation in a CD4-independent DNA vaccination model (17).

We here clarify these discrepant findings and demonstrate that agonist anti-CD27 mAb is synergistic with PD-1/L1 blockade in promoting CD8+ T-cell expansion, function, and tumor protection in multiple models. Functionally, we find that anti-CD27 and PD-1/L1 blockade cooperate to drive proliferative and cytotoxic gene expression programs in CD8+ T cells. These data reveal the complex interplay between costimulatory and inhibitory receptors in regulating T-cell activation and provide new impetus for evaluating combined agonist anti-CD27 and PD-1/L1 blockade in patients.

Cell lines and reagents

The cell lines B16-OVA-GFP, B16-BL6, FVAX, and C1498 have all been described previously (18–20) and were maintained in Dulbecco's modified Eagles Medium containing 10% FCS, pyruvate, l-glutamine, and antibiotics. Stocks of cells were maintained in liquid nitrogen. Defrosted cells were discarded within 3 months and regularly visualized for any changes in morphology or growth rate. Cell lines were routinely tested for mycoplasma contamination using endosafe-PTS cartridges (Charles River Laboratories). BCL1 tumor was maintained by passage in BALB/c mice as described (21). Endotoxin-low peptides hgp1002533 (KVPRNQDWL), OVA257–264 (SIINFEKL), and the altered peptide ligand SIIQFEKL were obtained from Peptide Protein Research Ltd. Cyclophosphamide and FTY720 were obtained from Sigma-Aldrich, and Cell Proliferation Dye eFluor450 and Fixable Viability Dye eFluor506 were from eBioscience. PE-labeled SIINFEKL peptide/H-2Kb tetramers were manufactured in house (Protein Core Facility). Antibodies used in vivo were rat anti-mouse CD27 (AT124; rat IgG2a), anti-OX40 (OX-86), anti-GITR (DTA-1), anti-4-1BB (LOB12.3), anti-CD4 (GK1.5 and YTA3.1.2), anti-CD8 (YTS169), rat IgG2a isotype control antibodies Mc106A5 or Mc39-16 (all prepared <10 EU/mg endotoxin in house); anti-mouse PD-1 (RMP1-14), anti-mouse PD-L1 (10F.9G2), and rat IgG2b isotype control mAbs were obtained from Bio X Cell. Anti-human CD27 mAb, varlilumab, has been described previously (21).

Mice and in vivo protocols

C57BL/6, BALB/c, OT-I, pmel1, and hCD27-Tg mice have been described previously (21, 22). Experiments were conducted according to UK Home Office Regulations or according to the guidelines established by the Institutional Animal Care and Use Committee (IACUC) at Celldex Therapeutics, Inc. For adoptive CD8+ T-cell transfer, single-cell suspensions of splenocytes were prepared in phosphate-buffered saline (PBS) and 1× 106 to × 106 CD8+Vβ13+Thy1.1+ cells (pmel1) or 1 × 104 CD8+SIINFEKL/H-2Kb tetramer+ (OT-I) cells were injected intravenously (i.v.). For tumor challenge, adherent cell lines were treated with trypsin-EDTA, washed, and viable cells were counted and injected into mice. C1498 cells were washed and counted prior to i.v. injection. BCL1 cells were maintained by in vivo passage, and hCD27-Tg mice were challenged with 1 × 107 cells i.v. For in vivo depletion experiments, mice received a total of 3 mg anti-CD4 antibody (a 50/50 mix of GK1.5 and YTA3.1.2) or a minimum total of 1.5 mg anti-CD8 (clone YTS169) split over three intraperitoneal (i.p.) injections between days −7 and +4 relative to tumor challenge. FVAX cells were treated with trypsin-EDTA, washed in PBS, and irradiated (208 Gy) prior to subcutaneous (s.c.) injection on the opposite flank to that of live B16-BL6 cells, on days 3, 6, and 9 relative to tumor challenge. For in vivo blockade of IL2, mice were treated i.p. on days 0, 1, and 2 relative to peptide vaccination, with a mix of anti-IL2 mAbs JES6-1A12 (50 μg/day; Bio X Cell) and S4B6.1 (200 μg/day; in house) as reported previously (23). For FTY720 treatment, mice were treated i.p. on alternate days with 50 μg drug in 200 μL PBS/2.5% DMSO prepared fresh, or with PBS/DMSO as a control.

Flow cytometry

Antibodies used for flow cytometry were anti-Thy1.1-APC or -eFluor450 (HIS51), anti-CD8α-FITC, -APC or -APC-Cy7 (53–6.7), anti-CD4-FITC, -eFluor450 or -APC (GK1.5), anti-OX40-PE (OX86), anti-CD27-PE (LG.3A10), anti-GITR-PE (DTA-1), anti–4-1BB-PE (17B5), anti–PD-1-APC (J43), anti-Vβ13-FITC (MR12.3), anti-CD107a-eFluor450 (eBio104B), anti-T-bet-FITC (eBio4B10), anti-IFNγ-APC (XMG1.2), anti-TNFα-FITC (MP6-XT22), anti-IL2-PE (JES6-5H4), anti-Foxp3-PE (FJK-16s), anti-CD25-PE (PC61.5), and appropriate isotype controls (all from eBioscience) as well as anti-granzyme B-APC (GB11; Invitrogen), anti-CD8-PE (YTS169; in house), anti–Myc-AlexaFluor647 (D84C12; Cell Signaling Technology), anti-Stat5(pY694)-AlexaFluor647 (47/Stat5; BD Biosciences) and appropriate isotype controls. For analysis of tumor-infiltrating lymphocytes (TIL), tumors were passed through a 70-μm mesh, and mononuclear cells were isolated by density gradient centrifugation prior to flow cytometric staining.

For all flow cytometry, cells were incubated with 10 μg/mL anti-Fc receptor mAb (2.4G2; in house) for 10 minutes prior to surface staining. For subsequent intracellular staining of T-bet, Foxp3, granzyme B, and Myc, cells were treated using the Foxp3/transcription factor buffer staining set (eBioscience). For detection of surface CD107a, splenocytes were restimulated for 4 hours with 1 μmol/L relevant or control peptide and either anti-CD107a-eF450 or an isotype control in the presence of Golgistop (BD Pharmingen) prior to resurface staining for CD107a and other surface markers as indicated. For the detection of intracellular cytokines, splenocytes were restimulated for 4 to 5 hours with 1 μmol/L relevant or control peptide in the presence of Golgiplug (BD Pharmingen) prior to surface staining, fixation in 1% formaldehyde/PBS and intracellular cytokine staining in 0.5% saponin/PBS. For the detection of intracellular Stat5(pY694), splenocytes were restimulated ex vivo with 20 U/mL IL2 for 15 minutes prior to surface staining for CD8 and Thy1.1, fixation (Cytofix/Cytoperm kit; BD Biosciences), treatment with ice-cold methanol for 30 minutes and intracellular staining for 30 minutes (Cytofix/Cytoperm kit). Flow cytometry was performed on a FACSCalibur using BD CellQuest software or on a FACSCantoII using BD FACSDiva software. FCS Express V3 was used for figure preparation.

Microarray analysis

For transcriptome analysis, mice received 2 × 106 pmel1 CD8+ T cells prior to i.v. injection of 100 μg hgp100 peptide and 400 μg total IgG comprised of 200 μg anti-CD27, 100 μg anti–PD-1, 100 μg anti–PD-L1, or isotype control mAbs to make up the total IgG dose. Splenocytes were collected on day 4 relative to immunization, CD8+ T cells were isolated by negative selection (CD8+ T-cell isolation kit; Miltenyi Biotech), and CD8+Thy1.1+ cells were positively sorted on a FACSAria to >99% purity. A minimum of 0.4 × 106 cells were collected, RNA was extracted using RNeasy kits and gDNA eliminator columns (QIAGEN) and stored at −80°C. Samples were analyzed on an Affymetrix Mouse Transcriptome Array 1.0 (AROS Applied Biotechnology). Raw microarray data were quantile-normalized using the Bioconductor R package “oligo.” Genes were tested for differential expression using an empirical Bayes moderated t test as implemented in the limma R package (version 3.30.9). The moderated t statistic was calculated for each gene and P values were corrected for multiple testing across genes and contrasts using the decideTests function (adjustment method = “separate”). The threshold for significant differential expression was a false discovery rate of q < 0.05. Subsequent data analysis used Ingenuity Pathway Analysis (IPA; QIAGEN).

Statistical analysis

Experimental statistical analyses were performed using GraphPad Prism software. Student two-tailed t test, log-rank (Mantel–Cox) test and two-way ANOVA (with Bonferroni post hoc test) were used throughout as indicated in the text. Data were considered statistically significant at P < 0.05.

Data availability

Experimental datasets generated during this study are available from the corresponding author upon reasonable request. Data generated from the microarray have been uploaded to the NCBI Gene Expression Omnibus and are available as GSE96923.

Anti-CD27 is superior to other anti-TNFRSF mAb for CD8+ T-cell expansion in vivo

With the ultimate aim of combining an effective TNF receptor superfamily (TNFRSF) agonist with PD-1 blockade, we initially compared several agonist anti-TNFRSF mAbs for their ability to augment CD8+ T-cell expansion. To this end, gp100-specific CD8+ T cells from pmel1 transgenic mice were adoptively transferred to congenic recipients prior to injection of peptide alone or with agonist mAb as indicated. Human gp100 peptide (hgp100) is approximately 100-fold more potent than murine gp100 in stimulating pmel1 CD8+ T cells (22, 24) and therefore hgp100 peptide was used for this, and all subsequent, experiments. Within the limited panel of mAbs evaluated, all mAbs are known T-cell agonists (12, 25, 26), yet in this setting only anti-CD27 mAb was able to significantly expand pmel1 CD8+ T cells compared with hgp100 peptide alone (Supplementary Fig. S1A). Analysis of TNFRSF receptor expression on CD8+ pmel1 T cells (Supplementary Fig. S1B) confirmed that resting CD8+ T cells express CD27 and GITR but not OX40 or 4-1BB, in line with previous publications (27, 28). OX40 and 4-1BB were both upregulated at 48 hours, but expression of these receptors was still relatively low compared with expression of CD27 and GITR at the same time point. Importantly, we noted that stimulation of pmel1 CD8+ T cells with peptide alone was sufficient to cause upregulation of the inhibitory PD-1 receptor and PD-1 remained on pmel1 cells after stimulation with peptide and anti-CD27 (Supplementary Fig. S1C).

Optimal CD8+ T-cell expansion and differentiation into effector cells require CD27 costimulation and PD-1/L1 blockade

To assess if PD-1 expression on activated CD8+ T cells limits the activity of agonist anti-CD27, we examined the effect of combining agonist anti-CD27 with blockade of the PD-1/L1 pathway on T-cell priming. Data shown in Fig. 1 reveal that the effects of combined treatment on pmel1 T-cell expansion are indeed synergistic. Thus, while T-cell proliferation, determined by cell-proliferation dye dilution, was induced by anti-CD27 and anti–PD-1/PD-L1, it was more extensive following the combination treatment (Fig 1A, top and bottom left). Enumerating pmel1 T cells in the spleen revealed that while anti-CD27 and anti–PD-1/L1 produced an increase in pmel1 cells of 17-fold and 6-fold, respectively, compared with peptide alone, the combination treatment resulted in a 64-fold increase in pmel1 CD8+ T-cell number (Fig. 1A, bottom right). The enhanced expansion afforded by the combination treatment was not limited to pmel1 T cells and was also evident after stimulation of OT-I transgenic CD8+ T cells with either high- or low-affinity ovalbumin-derived peptides (Supplementary Fig. S2A and S2B).

Figure 1.

Agonist anti-CD27 and PD-1/L1 blockade synergize to increase CD8+ T-cell expansion and effector function. A–D, Groups of 4 C57BL/6 mice were injected with 2×106 pmel1 CD8+Thy1.1+ cells on day −1, with hgp100 peptide and 200 μg each of anti-CD27, an equal mix of anti–PD-1 and anti–PD-L1, and/or isotype control antibodies to an equivalent final antibody dose on day 0 and the same antibody cocktails (minus peptide) on day 1. A, top, On day 3, the intensity of cell-proliferation dye in blood CD8+Thy1.1+ cells was determined from mice stimulated with peptide alone (filled gray), or with anti-CD27 (gray line), anti–PD-1/L1 mAbs (black line), or both anti-CD27 and anti–PD-1/L1 (thick black line). A, bottom left, Mean fluorescence intensity of cell-proliferation dye in CD8+Thy1.1+ cells on day 3 is shown. A, bottom right, The number of splenic CD8+Thy1.1+ cells was determined on day 4. B, Mean fluorescence intensity of T-bet and granzyme Bin ex vivo splenic CD8+Thy1.1+ cells and %CD107a+ of pmel1 cells after brief in vitro restimulation on day 4. C, Representative intracellular IFNγ, TNFα, and IL2 staining of CD8+Thy1.1+ cells, and % cytokine-positive of CD8+Thy1.1+ cells on day 4 after brief in vitro restimulation with peptide; lines as in A. D, Percentage of CD8+Thy1.1+ cells expressing combinations of IFNγ, TNFα, and IL2. Data in (A, left) are from one experiment; remaining data are from one of three similar experiments. Bar graphs in A–C show group means ± SEM. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.0001, two-tailed Student t test.

Figure 1.

Agonist anti-CD27 and PD-1/L1 blockade synergize to increase CD8+ T-cell expansion and effector function. A–D, Groups of 4 C57BL/6 mice were injected with 2×106 pmel1 CD8+Thy1.1+ cells on day −1, with hgp100 peptide and 200 μg each of anti-CD27, an equal mix of anti–PD-1 and anti–PD-L1, and/or isotype control antibodies to an equivalent final antibody dose on day 0 and the same antibody cocktails (minus peptide) on day 1. A, top, On day 3, the intensity of cell-proliferation dye in blood CD8+Thy1.1+ cells was determined from mice stimulated with peptide alone (filled gray), or with anti-CD27 (gray line), anti–PD-1/L1 mAbs (black line), or both anti-CD27 and anti–PD-1/L1 (thick black line). A, bottom left, Mean fluorescence intensity of cell-proliferation dye in CD8+Thy1.1+ cells on day 3 is shown. A, bottom right, The number of splenic CD8+Thy1.1+ cells was determined on day 4. B, Mean fluorescence intensity of T-bet and granzyme Bin ex vivo splenic CD8+Thy1.1+ cells and %CD107a+ of pmel1 cells after brief in vitro restimulation on day 4. C, Representative intracellular IFNγ, TNFα, and IL2 staining of CD8+Thy1.1+ cells, and % cytokine-positive of CD8+Thy1.1+ cells on day 4 after brief in vitro restimulation with peptide; lines as in A. D, Percentage of CD8+Thy1.1+ cells expressing combinations of IFNγ, TNFα, and IL2. Data in (A, left) are from one experiment; remaining data are from one of three similar experiments. Bar graphs in A–C show group means ± SEM. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.0001, two-tailed Student t test.

Close modal

Furthermore, combination treatment, but not anti-CD27 or anti–PD-1/L1 alone, increased expression of the T-box transcription factor T-bet (Fig. 1B), a key positive regulator of T-cell effector function and IFNγ synthesis (29). In addition, combination-activated CD8+ T cells revealed a more effector-like phenotype with significantly higher levels of intracellular granzyme B, surface CD107a, a surrogate marker of degranulation (Fig. 1B), and of intracellular IFNγ and TNFα (Fig. 1C) compared with cells treated with either anti-CD27 or anti–PD-1/PD-L1 alone. Consistent with previous findings by us and others in which IL2 transcription was identified as a major output of CD27 activation in T cells (15, 30), anti-CD27 significantly increased IL2 production compared with peptide alone, and this could not be further amplified by PD-1/L1 blockade (Fig. 1C). Finally, combined anti-CD27 and PD-1/L1 blockade also significantly increased the frequency of polyfunctional CD8+ T cells such that 41% of cells produced 2 or 3 cytokines after combination treatment compared with only 25% of anti-CD27–treated and <3% of anti–PD-1/L1–treated cells (Fig. 1D; P < 0.02, Student t test). Overall, these data demonstrate that many of the costimulatory effects of agonist anti-CD27 during priming are dampened by inhibitory PD-1 signaling and that the combination of PD-1 blockade and CD27 costimulation can massively boost CD8+ T-cell priming compared with either treatment alone.

Combined agonist anti-CD27 and PD-1 blockade activates a distinct gene expression program in CD8+ T cells

To investigate the mechanism(s) through which agonist anti-CD27 and PD-1/L1 blockade combine to enhance CD8+ T-cell expansion and function, adoptively transferred CD8+Thy1.1+ pmel1 cells were stimulated with peptide and mAb in vivo (either anti–PD-1/L1 or anti-CD27 alone or in combination) and purified at the peak of the response (Supplementary Fig. S3), prior to transcriptome analysis by microarray. Minimal expansion of pmel1 cells after injection of peptide alone precluded inclusion of this group. After normalization, two-dimensional unsupervised clustering was used to generate a heatmap showing gene-expression differences for the shared set of genes exhibiting significant changes in expression between both of the monotherapy groups and the combination-treated group (418 genes) and the smaller gene set (174 genes) whose expression was significantly changed between the highest-performing monotherapy (anti-CD27) and the combination group (Fig. 2A). We considered that these genes were most likely to be responsible for the synergistic effects of anti-CD27 and PD-1/L1 blockade given that anti-CD27 alone was the most effective of the monotherapies. The heatmap therefore represented 592 genes in which gene expression patterns tightly clustered into the three experimental groups, demonstrating minimal within-group variation.

Figure 2.

Anti-CD27 and PD-1/L1 blockade synergize to enhance CD8+ T-cell activation at a transcriptional level. Groups of 3 mice received 2×106 pmel1 CD8+Thy1.1+ T cells prior to injection with hgp100 peptide and anti-CD27, a mix of anti–PD-1 and anti–PD-L1, or all three mAbs combined. Four days later, RNA from purified splenic pmel1 CD8+ T cells was isolated and subjected to microarray analysis. A, Two-dimensional unsupervised clustering generated a heatmap representing log2 values of relative mRNA expression changes, and data clustered into 6 groups as indicated. B and C, The most prevalent functions of genes represented in clusters (B) 3 and (C) 4 were determined using IPA (bottom), and genes associated with the most highly significant process are represented diagrammatically (top). Depth of color represents the magnitude of gene-expression change from anti-CD27-stimulated to combination-stimulated cells (red upregulated and green downregulated); arrow/line color symbolizes activation (orange), a gene change that is inconsistent with the downstream molecule (yellow), or that the effect is not predicted (gray); symbol shape gives an indication of function. D, Mean ± SEM gene-expression values representing the frequencies of the gzma, gzmb, and gzmk transcripts in each group. B and C,P values taken from “Diseases and Functions” analysis in IPA calculated by Fisher exact test, (D) Benjamini–Hochberg adjusted Student t test: *, P < 0.05; **, P < 0.005 or as indicated.

Figure 2.

Anti-CD27 and PD-1/L1 blockade synergize to enhance CD8+ T-cell activation at a transcriptional level. Groups of 3 mice received 2×106 pmel1 CD8+Thy1.1+ T cells prior to injection with hgp100 peptide and anti-CD27, a mix of anti–PD-1 and anti–PD-L1, or all three mAbs combined. Four days later, RNA from purified splenic pmel1 CD8+ T cells was isolated and subjected to microarray analysis. A, Two-dimensional unsupervised clustering generated a heatmap representing log2 values of relative mRNA expression changes, and data clustered into 6 groups as indicated. B and C, The most prevalent functions of genes represented in clusters (B) 3 and (C) 4 were determined using IPA (bottom), and genes associated with the most highly significant process are represented diagrammatically (top). Depth of color represents the magnitude of gene-expression change from anti-CD27-stimulated to combination-stimulated cells (red upregulated and green downregulated); arrow/line color symbolizes activation (orange), a gene change that is inconsistent with the downstream molecule (yellow), or that the effect is not predicted (gray); symbol shape gives an indication of function. D, Mean ± SEM gene-expression values representing the frequencies of the gzma, gzmb, and gzmk transcripts in each group. B and C,P values taken from “Diseases and Functions” analysis in IPA calculated by Fisher exact test, (D) Benjamini–Hochberg adjusted Student t test: *, P < 0.05; **, P < 0.005 or as indicated.

Close modal

Six clusters of gene expression were observed, with clusters 3 and 4 representing genes upregulated in the combination group compared with either anti-CD27 or anti–PD-1/PD-L1 single treatment (Fig. 2A). These genes were either predominantly driven by anti-CD27 (cluster 3; 254 genes) or anti–PD-1/L1 (cluster 4; 120 genes), and overall both clusters were enriched for genes encoding proteins with a role in DNA replication and cell proliferation (Fig. 2B and C). Closer scrutiny (Supplementary Table S1) revealed that cluster 3 also included genes encoding the T-cell effector/memory differentiation transcription factors Id2 and Eomes as well as genes encoding enzymes driving glycolysis (Pgam1, Pfkp), glutaminolysis (Ppat), and fatty acid and cholesterol synthesis (Lss, Acaca, Fads1, Dhcr24). Cluster 4 also included genes involved in these metabolic pathways (e.g., Tpi1, Fads2, and Fdps), yet only cluster 4 and not cluster 3, additionally contained genes linked to cytotoxic function (P = 1.44 × 10−10, z = 1.764), including genes encoding granzymes A, B, and K and killer cell lectin-like receptors NKG2A, NKG2C, NKG2D, and NKG2E (Fig. 2C and Supplementary Tables S1 and S2). Furthermore, cluster 4 incorporated additional genes associated with effector CD8+ T cells including receptors for key cytokines (IL2 and IL12) and Tbx21, which encodes T-bet (Fig. 2C and Supplementary Table S2). Comparison of the genes in clusters 3 and 4, which exhibited the greatest fold change between single treatment and combination groups, also revealed a bias toward genes controlling cell proliferation and T-cell effector function in clusters 3 and 4, respectively (Supplementary Table S2). The top functional networks for each of clusters 3 and 4 (Fig. 2B and C, respectively) and normalized expression values for each of the granzymes in cluster 4 (Fig. 2D) are shown. Together, these data indicate that anti-CD27 and PD-1/L1 blockade cooperate to control a proproliferative and cytotoxic gene expression program that culminates in enhanced T-cell expansion and effector function.

Analysis of the transcription factors that promote expression of genes in clusters 3 and 4 revealed Myc and E2F1 to be highly significant regulators of both gene clusters (Fig. 3A–D). Myc is a key driver of T-cell proliferation and growth (31, 32) and E2F1 together with E2F2 control homeostatic T-cell proliferation (33). In support of a key role for Myc in facilitating CD8+ T-cell activation, combination-treated CD8+ T cells expressed higher levels of Myc protein, compared with monotherapy-treated, CD8+ T cells (Fig. 3E and F). Furthermore, previous reports have shown that maintenance of Myc in activated T cells is dependent on IL2 signaling and amino-acid uptake, such that expression of the IL2-receptor alpha chain CD25 and the prevalence of amino-acid transporter Slc7a5 transcripts correlate with Myc expression (34, 35). Our finding that CD25 protein, phosphorylation of the downstream-signaling moiety Stat5, and the prevalence of Slc7a5 transcripts are significantly elevated in cells treated with combined anti-CD27 and PD-1/L1 blockade treatment compared with monotherapy-treated cells (Fig. 4A–E), suggests that these factors could have combined to enhance Myc expression. Furthermore, in vivo blockade of IL2 significantly reduced the expansion of CD8+ T cells after combined anti-CD27 and PD-1/L1 blockade (Fig. 4F), and hindered acquisition of effector proteins as evidenced by almost complete abrogation of granzyme B expression and significant reductions in the production of IFNγ and TNFα (Fig. 4G–I).

Figure 3.

Agonist anti-CD27 and PD-1/L1 blockade synergize for increased Myc activity. Positive upstream regulators of genes represented in clusters (A) 3 and (B) 4 as identified by IPA; P values (crosses; right axis) and z-scores (line; left axis) are indicated. Myc and/or E2F1-regulated genes in clusters (C) 3 and (D) 4 are shown with symbols and colors as in Fig. 2. E–F, Groups of mice received pmel1 CD8+ T cells prior to injection of hgp100 peptide and isotype control antibodies, anti-CD27, anti–PD-1, and anti–PD-L1, or with all three antibodies combined. On (E) day 2, or (F) the days indicated, the expression of intracellular Myc in splenic Thy1.1+ CD8+ cells was assessed by flow cytometry. E, Representative staining (black line) for Myc on Thy1.1+CD8+ cells stimulated in vivo as indicated; isotype control staining is shown in each case (gray). F, Mean (±SEM) % of Myc+ pmel1 CD8+ T cells at each time point combined from 3 independent experiments, each representing one time point. A and B,P values calculated by Fisher exact test performed by IPA. F, Two-way ANOVA with Bonferroni post hoc test; *, P < 0.01; ****, P < 0.0001.

Figure 3.

Agonist anti-CD27 and PD-1/L1 blockade synergize for increased Myc activity. Positive upstream regulators of genes represented in clusters (A) 3 and (B) 4 as identified by IPA; P values (crosses; right axis) and z-scores (line; left axis) are indicated. Myc and/or E2F1-regulated genes in clusters (C) 3 and (D) 4 are shown with symbols and colors as in Fig. 2. E–F, Groups of mice received pmel1 CD8+ T cells prior to injection of hgp100 peptide and isotype control antibodies, anti-CD27, anti–PD-1, and anti–PD-L1, or with all three antibodies combined. On (E) day 2, or (F) the days indicated, the expression of intracellular Myc in splenic Thy1.1+ CD8+ cells was assessed by flow cytometry. E, Representative staining (black line) for Myc on Thy1.1+CD8+ cells stimulated in vivo as indicated; isotype control staining is shown in each case (gray). F, Mean (±SEM) % of Myc+ pmel1 CD8+ T cells at each time point combined from 3 independent experiments, each representing one time point. A and B,P values calculated by Fisher exact test performed by IPA. F, Two-way ANOVA with Bonferroni post hoc test; *, P < 0.01; ****, P < 0.0001.

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Figure 4.

Maximal CD8+ T-cell activation by agonist anti-CD27 and PD-1/L1 blockade is dependent on IL2. A–D, Groups of mice received pmel1 CD8+ T cells prior to injection of hgp100 peptide and isotype control antibodies, anti-CD27, anti–PD-1, and anti–PD-L1, or with all three antibodies combined. On day (A) 3, (C and D) 4, or (B) the days indicated, the expression of (A and B) surface CD25 or (C and D) intracellular Stat5(pY694) on splenic Thy1.1+ CD8+ cells was assessed by flow cytometry. Representative staining (black line) for (A) CD25 and (C) Stat5(pY694) on Thy1.1+CD8+ cells stimulated in vivo as indicated; isotype control staining is shown in each case (gray). Mean (±SEM) % of (B) CD25+ and (D) Stat5(pY694)+ pmel1 CD8+ T cells. E, Slc7a5 transcript expression from the microarray experiment shown in Fig. 2. F–I, Groups of mice received Thy1.1+pmel1 CD8+ T cells and antibodies as (A–D) alone (no IL2 block) or were concurrently treated with IL2 blocking mAbs given starting 2 hours prior to peptide/activating mAb injection (day 0) and then given additionally on days 1 and 2. F, The frequency of splenic Thy1.1+CD8+ T cells on day 4 and (G) the mean fluorescence intensity of intracellular granzyme B staining ex vivo were determined by flow cytometry. H and I, The % of Thy1.1+CD8+ T cells accumulating intracellular IFNγ and TNFα was also assessed after brief in vitro restimulation. B, Data are combined from 3 independent experiments, each representing one time point. Two-way ANOVA with Bonferroni post hoc test ****, P < 0.0001. Data in C, D, and F–I are from one experiment with 4 mice per group. Student two-tailed t test; n.s., P > 0.05; *, P < 0.05; **, P < 0.005; ****, P < 0.0001. E, Benjamini–Hochberg adjusted Student t test: *, P < 0.05; ****, P < 0.0001.

Figure 4.

Maximal CD8+ T-cell activation by agonist anti-CD27 and PD-1/L1 blockade is dependent on IL2. A–D, Groups of mice received pmel1 CD8+ T cells prior to injection of hgp100 peptide and isotype control antibodies, anti-CD27, anti–PD-1, and anti–PD-L1, or with all three antibodies combined. On day (A) 3, (C and D) 4, or (B) the days indicated, the expression of (A and B) surface CD25 or (C and D) intracellular Stat5(pY694) on splenic Thy1.1+ CD8+ cells was assessed by flow cytometry. Representative staining (black line) for (A) CD25 and (C) Stat5(pY694) on Thy1.1+CD8+ cells stimulated in vivo as indicated; isotype control staining is shown in each case (gray). Mean (±SEM) % of (B) CD25+ and (D) Stat5(pY694)+ pmel1 CD8+ T cells. E, Slc7a5 transcript expression from the microarray experiment shown in Fig. 2. F–I, Groups of mice received Thy1.1+pmel1 CD8+ T cells and antibodies as (A–D) alone (no IL2 block) or were concurrently treated with IL2 blocking mAbs given starting 2 hours prior to peptide/activating mAb injection (day 0) and then given additionally on days 1 and 2. F, The frequency of splenic Thy1.1+CD8+ T cells on day 4 and (G) the mean fluorescence intensity of intracellular granzyme B staining ex vivo were determined by flow cytometry. H and I, The % of Thy1.1+CD8+ T cells accumulating intracellular IFNγ and TNFα was also assessed after brief in vitro restimulation. B, Data are combined from 3 independent experiments, each representing one time point. Two-way ANOVA with Bonferroni post hoc test ****, P < 0.0001. Data in C, D, and F–I are from one experiment with 4 mice per group. Student two-tailed t test; n.s., P > 0.05; *, P < 0.05; **, P < 0.005; ****, P < 0.0001. E, Benjamini–Hochberg adjusted Student t test: *, P < 0.05; ****, P < 0.0001.

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Gene expression changes in the remaining clusters of the heatmap were also largely consistent with the superior effector phenotype of CD8+ T cells treated with combination therapy (Supplementary Table S1). For instance, combination-induced genes in cluster 2 that showed increased expression when compared with anti-CD27 and comparable expression to anti–PD-1/L1, included the proeffector CD8+ T-cell gene, Rheb (36), and Ctla2a and Ctla2b, putative cysteine protease-like molecules (37). Similarly, in cluster 1, several T-cell inhibitory genes exhibited decreased expression after combination treatment compared with either monotherapy treatment, e.g., Rgs10, which limits Vav1-Rac1 activation and T-cell adhesion (38), Itm2c, a mediator of cell death downstream of TNFα (39) and Spry1, an inhibitor of CD8+ T-cell cytotoxicity (40). Furthermore, expression of Id3, which encodes a fate-determining transcription factor downregulated in effector CD8+ T cells (41), was also decreased in combination-treated CD8+ T cells in cluster 1.

A large number of genes in the heatmap (183/592) showed greatest expression after anti-CD27 treatment alone. For this gene cohort, inclusion of PD-1/L1 blockade inhibited expression to a level between that induced by each monotherapy (cluster 5), or to a baseline lower than either monotherapy (cluster 6). Consistent with an intermediate expression profile by combination-treated CD8+ T cells, cluster 5 genes included those with both positive (e.g., Rel, Lef1; refs. 42, 43) and negative (e.g., TNFAIP3, CD274; refs. 1, 44) influences on effector CD8+ T-cell proliferation and/or function. Genes represented in cluster 6 were diverse in function yet included Mir15b, Rgcc, and Ctla4, all inhibitors of T-cell cytokine production and/or proliferation (45, 46), consistent with their preferential suppression in combination-treated CD8+ T cells. A full list of the genes represented in each cluster can be found in Supplementary Table S1.

Agonist anti-CD27 and PD-1/L1 blockade synergize for improved adoptive T-cell therapy (ACT)

To ascertain whether the increase in CD8+ T-cell frequency and effector function seen after combined anti-CD27 and PD-1 blockade treatment translates into increased control of tumor, mice were challenged with a lethal dose of B16-BL6 melanoma cells and tumor allowed to establish prior to ACT of pmel1 CD8+ T cells and vaccination with peptide alone, with anti-CD27, anti–PD-1/L1 or with all three mAbs together. While neither anti-CD27 nor anti–PD-1/L1 had any substantial impact on tumor growth, combining anti-CD27 and anti–PD-1/L1 delayed tumor growth and significantly improved long-term survival (Fig. 5A and B). Experiments in which anti–PD-1 was used in place of the anti–PD-1/L1 cocktail similarly showed synergy between anti–PD-1 and anti-CD27 for tumor therapy (Supplementary Fig. S4A and S4B). Furthermore, the benefit of combined anti-CD27 and PD-1 blockade was only apparent when mAbs were delivered with ACT, confirming the CD8+ T-cell–dependent nature of the treatment (Supplementary Fig. S4C).

Figure 5.

Agonist anti-CD27 and PD-1/L1 blockade synergize for improved adoptive T-cell therapy. A and B, Groups of 5 C57BL/6 mice were challenged with 2×105 B16-BL6 tumor cells and received 3×106 CD8+Thy1.1+ cells from pmel1 mice 5 days later. Mice were injected the following day with 200 μg hgp100 peptide delivered with anti-CD27, anti–PD-1, and anti–PD-L1, with all three antibodies together (combination) or with isotype control mAb and were boosted with these same antibodies a day later. Mice were culled when mean tumor diameter reached 15 mm. Data are pooled from 2 experiments. A, Mean tumor diameter in each group; B, % survival over time. A, *, P < 0.05 two-way ANOVA; B, **, P < 0.005 log-rank (Mantel–Cox) test.

Figure 5.

Agonist anti-CD27 and PD-1/L1 blockade synergize for improved adoptive T-cell therapy. A and B, Groups of 5 C57BL/6 mice were challenged with 2×105 B16-BL6 tumor cells and received 3×106 CD8+Thy1.1+ cells from pmel1 mice 5 days later. Mice were injected the following day with 200 μg hgp100 peptide delivered with anti-CD27, anti–PD-1, and anti–PD-L1, with all three antibodies together (combination) or with isotype control mAb and were boosted with these same antibodies a day later. Mice were culled when mean tumor diameter reached 15 mm. Data are pooled from 2 experiments. A, Mean tumor diameter in each group; B, % survival over time. A, *, P < 0.05 two-way ANOVA; B, **, P < 0.005 log-rank (Mantel–Cox) test.

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Agonist anti-CD27 and PD-1/L1 blockade synergize for enhanced endogenous antitumor CD8+ T-cell responses

We next sought to test whether this dual-targeting strategy could promote endogenous antitumor immunity. To this end, mice were challenged with the more immunogenic B16-OVA-GFP tumor prior to mAb injection, in the absence of ACT. Again, anti-CD27 and PD-L1 blockade together synergized to impair tumor growth and significantly improve long-term survival, in marked contrast to monotherapy with either anti-CD27 or anti–PD-L1, which alone had little impact on survival (Fig. 6A and B). Therapy with combined anti-CD27 and anti–PD-L1 was entirely dependent on endogenous CD8+ T cells, but not on CD4+ T cells (Fig. 6C), consistent with an increased frequency of CD8+ T cells in the tumor after combination treatment (Fig. 6D). Even in an aggressive model of melanoma in which treatment needs to be combined with a cellular vaccine, the combination of anti-CD27 and anti–PD-L1, but neither agent alone, significantly retarded tumor growth (Supplementary Fig. S5A) and synergized to enhance accumulation of local CD8+ T cells (Supplementary Fig. S5B). Furthermore, blocking lymphocyte egress from lymph nodes in mice bearing small established tumors had no detrimental effect on tumor therapy driven by anti-CD27 and PD-1/L1 blockade, suggesting direct activation of tumor-resident T cells (Fig. 6E and F). To determine whether the synergy between anti-CD27 and PD-1 blockade was applicable to tumor types other than melanoma, we also evaluated this approach in a model of acute myeloid leukemia. Similarly, only the combination of anti-CD27 and PD-1 blockade, and neither therapy alone, conferred significant therapeutic benefit (Fig. 6G). A similar trend toward improved tumor protection by the combination treatment was also observed when mice were treated with mAb in conjunction with chemotherapy (Fig. 6H).

Figure 6.

Agonist anti-CD27 and PD-1/L1 blockade synergize for enhanced endogenous antitumor CD8+ T-cell responses in wild-type and hCD27-Tg mice. A–F, Groups of mice were challenged with 5×105 B16-OVA-GFP tumor cells s.c. prior to treatment with (A–C) 200 μg anti-CD27, anti–PD-L1, a combination of the two, or with isotype control mAb delivered i.p. on days 5, 7, and 9. C, Groups of mice additionally received depleting antibodies to CD4 or CD8. D, Frequency of CD8+ T cells within live lymphocytes in B16-OVA-GFP tumor after treatment with the mAbs indicated on days 10, 12, and 14; mice were culled on day 19. In E and F, mice were treated with a mix of anti-CD27, anti–PD-1, and anti–PD-L1 or received isotype control antibodies on days 7, 9, and 11 and received i.p. injections of FTY720, or control, every other day from day 6 to 24 inclusive. Data in E show percentage of CD8+ (left) and CD4+ (right) cells of lymphocytes in the blood on day 7 prior to mAb injection. Data in F show mean tumor growth in each group. G and H, Groups of mice were challenged with 106 C1498 cells i.v. prior to injection of (G) 200 μg anti–PD-1, anti-CD27, both together, or control antibodies every other day from days 10 to 20 inclusive, or (H) 180 mg/kg cyclophosphamide and injection of 150 μg antibodies on days 8, 10, 11, 13, and 15. I, Groups of 10 hCD27-Tg mice were challenged with BCL1 tumor cells prior to i.p. injection of 200 μg anti-human CD27 on days 4, 6, 8, 10, and 12 and/or 100 μg anti–PD-L1 on days 4, 6, and 8. Data in B, C, and G–I show % survival to the humane endpoint, and data are pooled from (A, B, G, and I) 3, (C) 2, or (D–F and H) 1 independent experiment(s). A and F, *, P < 0.05; **, P < 0.005, two-way ANOVA; B, C, and G–I; **, P < 0.005; ****, P < 0.0001, or as indicated, log-rank (Mantel–Cox) test; D and E, **, P < 0.005; ***, P < 0.0005, Student two-tailed t test.

Figure 6.

Agonist anti-CD27 and PD-1/L1 blockade synergize for enhanced endogenous antitumor CD8+ T-cell responses in wild-type and hCD27-Tg mice. A–F, Groups of mice were challenged with 5×105 B16-OVA-GFP tumor cells s.c. prior to treatment with (A–C) 200 μg anti-CD27, anti–PD-L1, a combination of the two, or with isotype control mAb delivered i.p. on days 5, 7, and 9. C, Groups of mice additionally received depleting antibodies to CD4 or CD8. D, Frequency of CD8+ T cells within live lymphocytes in B16-OVA-GFP tumor after treatment with the mAbs indicated on days 10, 12, and 14; mice were culled on day 19. In E and F, mice were treated with a mix of anti-CD27, anti–PD-1, and anti–PD-L1 or received isotype control antibodies on days 7, 9, and 11 and received i.p. injections of FTY720, or control, every other day from day 6 to 24 inclusive. Data in E show percentage of CD8+ (left) and CD4+ (right) cells of lymphocytes in the blood on day 7 prior to mAb injection. Data in F show mean tumor growth in each group. G and H, Groups of mice were challenged with 106 C1498 cells i.v. prior to injection of (G) 200 μg anti–PD-1, anti-CD27, both together, or control antibodies every other day from days 10 to 20 inclusive, or (H) 180 mg/kg cyclophosphamide and injection of 150 μg antibodies on days 8, 10, 11, 13, and 15. I, Groups of 10 hCD27-Tg mice were challenged with BCL1 tumor cells prior to i.p. injection of 200 μg anti-human CD27 on days 4, 6, 8, 10, and 12 and/or 100 μg anti–PD-L1 on days 4, 6, and 8. Data in B, C, and G–I show % survival to the humane endpoint, and data are pooled from (A, B, G, and I) 3, (C) 2, or (D–F and H) 1 independent experiment(s). A and F, *, P < 0.05; **, P < 0.005, two-way ANOVA; B, C, and G–I; **, P < 0.005; ****, P < 0.0001, or as indicated, log-rank (Mantel–Cox) test; D and E, **, P < 0.005; ***, P < 0.0005, Student two-tailed t test.

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Agonist anti-human CD27 and PD-1/L1 blockade synergize for improved tumor therapy in human CD27 transgenic mice

Finally, to move toward clinical translation, we evaluated whether a previously described, and clinically relevant, agonistic anti-human CD27 mAb, varlilumab (21), also synergizes with PD-L1 blockade in vivo. Human CD27 transgenic mice (21) were challenged with a lethal dose of BCL1 lymphoma cells prior to injection with varlilumab, anti–PD-L1, or both mAb together in a suboptimized setting in which varlilumab alone does not show efficacy. In line with our findings across multiple established tumor models in wild-type mice, treatment of human CD27 transgenic (hCD27-Tg) mice with anti-human CD27 mAb or with anti–PD-L1 alone had little impact on tumor growth, yet combined treatment conferred significant long-term protection with over half of the mice surviving longer than 80 days (Fig. 6I).

Blockade of the PD-1–PD-L1 pathway in humans has produced significant benefits across multiple types of solid tumors, but response rates remain modest and vary from ∼15% to 40% depending on the tumor type (47). Recent data indicate that resistance to PD-1 immunotherapy in the nonresponding subset is not due to a lack of antigens, but correlates with the absence of CD8+ T cells within the tumor (48). This suggests that strategies that boost the number of infiltrating T cells, for example through augmenting T-cell priming, could improve the therapeutic response of anti–PD-1/L1 mAb. In this study, we have started to address this premise by investigating if the combination of agonist anti-CD27 mAb could synergize with blockade of the PD-1 pathway for improved antitumor immunity. CD27 is a well-characterized costimulatory receptor for CD8+ T cells, and when tested as a monotherapy, agonist anti-CD27 mAb was superior to mAbs targeting other costimulatory receptors such as OX40, 4-1BB, and GITR in driving expansion of gp100-specific CD8+ T cells in vivo (Supplementary Fig. S1). We show here that the combination of agonist anti-CD27 and anti–PD-1/L1 mAb is indeed synergistic; the combined treatment resulted in improved priming of pmel1 CD8+ T cells as evidenced by increased T-cell proliferation and enhanced differentiation into effector T cells (Fig. 1). Consequently, when compared with monotherapy, the combined treatment afforded more robust antitumor immunity in a number of preclinical tumor models. Furthermore, the observed synergy was not limited to a single anti-CD27 mAb because varlilumab, an anti-human CD27 mAb currently in phase I/II clinical testing, also successfully synergized with PD-1 blockade in augmenting antitumor immunity in mice that expressed human CD27.

By characterizing the transcriptomes of pmel1 CD8+ T cells primed under different conditions in vivo, we found that anti-CD27 mAb and blockade of PD-1 synergized by reinforcing the expression of genes that regulate cell proliferation and those associated with cytotoxicity of CD8+ T cells. Unexpectedly, the contribution of CD27 triggering and PD-1 blockade was not equivalent with CD27 costimulation exerting a more dominant role in regulating the proliferation program (Fig. 2 and Supplementary Table S2). Thus, among the 592 genes that were significantly changed in the combination arm compared with each of the monotherapy arms, 114 (19.2%) cell proliferation–associated genes were induced strongly by CD27 costimulation (Fig. 2 and Supplementary Tables S1 and S2). In contrast, only 57 genes (9.6%) associated with cell proliferation were differentially induced by PD-1 blockade. We identified Myc as a potential driver of a significant number of genes upregulated by the combination treatment (Fig. 3A–D). Previous reports have shown that the expression of Myc regulates metabolic reprogramming of T cells (31), correlates with expression of IFNγ and CD69 (34), and regulates the extent of cell proliferation with small changes in Myc resulting in large changes in cell frequency (32). Our data further show that anti-CD27 and PD-1/L1 blockade converge to maximally promote Myc protein levels and a Myc-regulated program of gene expression in vivo. Myc protein has a half-life of around 20 minutes in vivo but can be maintained by IL2 in a dose-dependent manner such that expression of CD25 correlates with Myc protein levels in T cells (34). Interestingly, our data show CD25 and phosphorylated Stat5 (pY694), a key downstream mediator of IL2 signaling, to be synergistically increased by anti-CD27 and PD-1/L1 blockade, providing a potential mechanism for cooperative maintenance of Myc by combination treatment, and suggesting that while anti-CD27 drives an increase in IL2, both anti-CD27 and PD-1 blockade may be required to maximize its capture and downstream signaling. IL2 maintains Myc predominantly at a posttranscriptional level dependent on amino-acid uptake via Slc7a5, itself upregulated by IL2 (34, 35). Consistent with cooperatively upregulated IL2 signaling, we find that anti-CD27 and PD-1/L1 blockade synergistically increases the frequency of Slc7a5 mRNA transcripts, further supporting an IL2-driven mechanism of Myc maintenance. Furthermore, blockade of IL2 significantly hindered the expansion of CD8+ T cells and their acquisition of effector functions, thus supporting our hypothesis that anti-CD27 and PD-1/L1 blockade converges to enhance proliferative and effector differentiation programs downstream of IL2 and Myc. However, confirmation of the role of IL2/Myc in this setting would require in-depth analysis of gene expression at a range of time points, particularly given that maximal Myc expression appears to precede maximal expression of CD25 (Figs. 3 and 4).

That we find anti-CD27 and anti–PD-1/L1 to synergize for CD8+ T-cell activation may reflect the influence of PD-1 and CD27 on largely distinct signaling pathways. CD27 uses TRAFs 2 and 5 to engage downstream JNK, and canonical and noncanonical NF-κB pathways leading to high levels of IL2 transcription (30, 49). In contrast, PD-1 recruits phosphatases to inhibit CD28 and possibly CD3ζ/ZAP70 phosphorylation, limit activation of the PI3K/Akt and Ras/MEK/Erk pathways and upregulate the transcription factor BATF to repress proliferation and IL2 production (1, 2, 50, 51). Interestingly, PD-1 inhibition synergizes with IL2 to reinvigorate exhausted T cells during chronic viral infection, suggesting that derepression of IL2 by PD-1 blockade elicits only suboptimal concentrations of this cytokine, at least in exhausted T cells (52). Our data similarly show that PD-1 blockade during priming has little effect on IL2 production by CD8+ T cells, suggesting that the ability of anti-CD27 to promote IL2 might be key to its effective cooperation with anti–PD-1/L1.

Together, our data provide clear evidence that agonist anti-CD27 antibodies combine successfully with PD-1/L1 blockade to improve CD8+ T-cell activation, frequency, and tumor therapy in a range of settings, and support the ongoing evaluation of agonist anti-CD27 (varlilumab) with PD-1/L1 blockade in patients (NCT02335918). Of note, a recent report has shown that the extent of T-cell reinvigoration following anti–PD-1 treatment determines the likelihood of clinical response in melanoma patients (53), suggesting that combining anti-CD27 mAb with PD-1 blockade may also increase the frequency of responders in the clinic. Finally, by defining the mechanism through which CD27 agonism and PD-1 blockade combine for optimal T-cell activation, our work also provides a platform to define biomarkers that predict clinical responses to immunotherapy.

L.J. Thomas, L.-Z. He, and T. Keler are employees of Celldex Therapeutics. S.L. Buchan has received salary support from Celldex Therapeutics. A. Al-Shamkhani has received grant funding from Celldex Therapeutics and is co-inventor on a patent currently licensed to that company. No potential conflicts of interest were disclosed by the other authors.

Conception and design: S.L. Buchan, S.M. Thirdborough, T. Keler, A. Al-Shamkhani

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S.L. Buchan, M. Fallatah, V.Y. Taraban, A. Rogel, L.J. Thomas, C.A. Penfold

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S.L. Buchan, M. Fallatah, S.M. Thirdborough, V.Y. Taraban, A. Rogel, L.-Z. He, A. Al-Shamkhani

Writing, review, and/or revision of the manuscript: S.L. Buchan, S.M. Thirdborough, A. Rogel, M.A. Curran, T. Keler, A. Al-Shamkhani

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S.L. Buchan, M. Fallatah, A. Al-Shamkhani

Study supervision: A. Al-Shamkhani

The authors thank members of the Biomedical Research Facility for animal husbandry, L. Douglas and P. Duriez of the Cancer Research UK (CRUK) Protein Production Facility for the PE-labeled H-2Kb/SIINFEKL tetramer, T. Inzhelevskaya for supplying antibodies for in vivo use, and Cancer Research UK for funding.

This work was also funded by Celldex Therapeutics.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Pauken
KE
,
Wherry
EJ
. 
Overcoming T cell exhaustion in infection and cancer
.
Trends Immunol
2015
;
36
:
265
76
.
2.
Hui
E
,
Cheung
J
,
Zhu
J
,
Su
X
,
Taylor
MJ
,
Wallweber
HA
, et al
T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition
.
Science
2017
;
355
:
1428
33
.
3.
Brown
KE
,
Freeman
GJ
,
Wherry
EJ
,
Sharpe
AH
. 
Role of PD-1 in regulating acute infections
.
Curr Opin Immunol
2010
;
22
:
397
401
.
4.
Fuse
S
,
Tsai
CY
,
Molloy
MJ
,
Allie
SR
,
Zhang
W
,
Yagita
H
, et al
Recall responses by helpless memory CD8+ T cells are restricted by the up-regulation of PD-1
.
J Immunol
2009
;
182
:
4244
54
.
5.
Gros
A
,
Robbins
PF
,
Yao
X
,
Li
YF
,
Turcotte
S
,
Tran
E
, et al
PD-1 identifies the patient-specific CD8(+) tumor-reactive repertoire infiltrating human tumors
.
J Clin Invest
2014
;
124
:
2246
59
.
6.
Chen
L
,
Han
X
. 
Anti-PD-1/PD-L1 therapy of human cancer: past, present, and future
.
J Clin Invest
2015
;
125
:
3384
91
.
7.
Blank
CU
,
Haanen
JB
,
Ribas
A
,
Schumacher
TN
. 
CANCER IMMUNOLOGY. The "cancer immunogram"
.
Science
2016
;
352
:
658
60
.
8.
Corrales
L
,
Matson
V
,
Flood
B
,
Spranger
S
,
Gajewski
TF
. 
Innate immune signaling and regulation in cancer immunotherapy
.
Cell Res
2017
;
27
:
96
108
.
9.
Taraban
VY
,
Rowley
TF
,
Al-Shamkhani
A
. 
Cutting edge: a critical role for CD70 in CD8 T cell priming by CD40-licensed APCs
.
J Immunol
2004
;
173
:
6542
6
.
10.
Taraban
VY
,
Rowley
TF
,
Tough
DF
,
Al-Shamkhani
A
. 
Requirement for CD70 in CD4+ Th cell-dependent and innate receptor-mediated CD8+ T cell priming
.
J Immunol
2006
;
177
:
2969
75
.
11.
Taraban
VY
,
Martin
S
,
Attfield
KE
,
Glennie
MJ
,
Elliott
T
,
Elewaut
D
, et al
Invariant NKT cells promote CD8+ cytotoxic T cell responses by inducing CD70 expression on dendritic cells
.
J Immunol
2008
;
180
:
4615
20
.
12.
French
RR
,
Taraban
VY
,
Crowther
GR
,
Rowley
TF
,
Gray
JC
,
Johnson
PW
, et al
Eradication of lymphoma by CD8 T cells following anti-CD40 monoclonal antibody therapy is critically dependent on CD27 costimulation
.
Blood
2007
;
109
:
4810
5
.
13.
Bak
SP
,
Barnkob
MS
,
Bai
A
,
Higham
EM
,
Wittrup
KD
,
Chen
J
. 
Differential requirement for CD70 and CD80/CD86 in dendritic cell-mediated activation of tumor-tolerized CD8 T cells
.
J Immunol
2012
;
189
:
1708
16
.
14.
Taraban
VY
,
Rowley
TF
,
Kerr
JP
,
Willoughby
JE
,
Johnson
PM
,
Al-Shamkhani
A
, et al
CD27 costimulation contributes substantially to the expansion of functional memory CD8(+) T cells after peptide immunization
.
Eur J Immunol
2013
;
43
:
3314
23
.
15.
Rowley
TF
,
Al-Shamkhani
A
. 
Stimulation by soluble CD70 promotes strong primary and secondary CD8+ cytotoxic T cell responses in vivo
.
J Immunol
2004
;
172
:
6039
46
.
16.
Buchan
SL
,
Manzo
T
,
Flutter
B
,
Rogel
A
,
Edwards
N
,
Zhang
L
, et al
OX40- and CD27-mediated costimulation synergizes with anti-PD-L1 blockade by forcing exhausted CD8+ T cells to exit quiescence
.
J Immunol
2015
;
194
:
125
33
.
17.
Ahrends
T
,
Babala
N
,
Xiao
Y
,
Yagita
H
,
van Eenennaam
H
,
Borst
J
. 
CD27 Agonism Plus PD-1 Blockade Recapitulates CD4+ T-cell Help in Therapeutic Anticancer Vaccination
.
Cancer Res
2016
;
76
:
2921
31
.
18.
Sancho
D
,
Mourao-Sa
D
,
Joffre
OP
,
Schulz
O
,
Rogers
NC
,
Pennington
DJ
, et al
Tumor therapy in mice via antigen targeting to a novel, DC-restricted C-type lectin
.
J Clin Invest
2008
;
118
:
2098
110
.
19.
Curran
MA
,
Montalvo
W
,
Yagita
H
,
Allison
JP
. 
PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors
.
Proc Natl Acad Sci U S A
2010
;
107
:
4275
80
.
20.
Boyer
MW
,
Orchard
PJ
,
Gorden
KB
,
Anderson
PM
,
McLvor
RS
,
Blazar
BR
. 
Dependency on intercellular adhesion molecule recognition and local interleukin-2 provision in generation of an in vivo CD8+ T-cell immune response to murine myeloid leukemia
.
Blood
1995
;
85
:
2498
506
.
21.
He
LZ
,
Prostak
N
,
Thomas
LJ
,
Vitale
L
,
Weidlick
J
,
Crocker
A
, et al
Agonist anti-human CD27 monoclonal antibody induces T cell activation and tumor immunity in human CD27-transgenic mice
.
J Immunol
2013
;
191
:
4174
83
.
22.
Overwijk
WW
,
Theoret
MR
,
Finkelstein
SE
,
Surman
DR
,
de Jong
LA
,
Vyth-Dreese
FA
, et al
Tumor regression and autoimmunity after reversal of a functionally tolerant state of self-reactive CD8+ T cells
.
J Exp Med
2003
;
198
:
569
80
.
23.
Willoughby
JE
,
Kerr
JP
,
Rogel
A
,
Taraban
VY
,
Buchan
SL
,
Johnson
PW
, et al
Differential impact of CD27 and 4-1BB costimulation on effector and memory CD8 T cell generation following peptide immunization
.
J Immunol
2014
;
193
:
244
51
.
24.
Overwijk
WW
,
Tsung
A
,
Irvine
KR
,
Parkhurst
MR
,
Goletz
TJ
,
Tsung
K
, et al
gp100/pmel 17 is a murine tumor rejection antigen: induction of "self"-reactive, tumoricidal T cells using high-affinity, altered peptide ligand
.
J Exp Med
1998
;
188
:
277
86
.
25.
Taraban
VY
,
Rowley
TF
,
O'Brien
L
,
Chan
HT
,
Haswell
LE
,
Green
MH
, et al
Expression and costimulatory effects of the TNF receptor superfamily members CD134 (OX40) and CD137 (4-1BB), and their role in the generation of anti-tumor immune responses
.
Eur J Immunol
2002
;
32
:
3617
27
.
26.
Lin
GH
,
Snell
LM
,
Wortzman
ME
,
Clouthier
DL
,
Watts
TH
. 
GITR-dependent regulation of 4-1BB expression: implications for T cell memory and anti-4-1BB-induced pathology
.
J Immunol
2013
;
190
:
4627
39
.
27.
Snell
LM
,
Lin
GH
,
Watts
TH
. 
IL-15-dependent upregulation of GITR on CD8 memory phenotype T cells in the bone marrow relative to spleen and lymph node suggests the bone marrow as a site of superior bioavailability of IL-15
.
J Immunol
2012
;
188
:
5915
23
.
28.
Croft
M
,
Duan
W
,
Choi
H
,
Eun
SY
,
Madireddi
S
,
Mehta
A
. 
TNF superfamily in inflammatory disease: translating basic insights
.
Trends Immunol
2012
;
33
:
144
52
.
29.
Sullivan
BM
,
Juedes
A
,
Szabo
SJ
,
von Herrath
M
,
Glimcher
LH
. 
Antigen-driven effector CD8 T cell function regulated by T-bet
.
Proc Natl Acad Sci U S A
2003
;
100
:
15818
23
.
30.
Peperzak
V
,
Xiao
Y
,
Veraar
EA
,
Borst
J
. 
CD27 sustains survival of CTLs in virus-infected nonlymphoid tissue in mice by inducing autocrine IL-2 production
.
J Clin Invest
2010
;
120
:
168
78
.
31.
Wang
R
,
Dillon
CP
,
Shi
LZ
,
Milasta
S
,
Carter
R
,
Finkelstein
D
, et al
The transcription factor Myc controls metabolic reprogramming upon T lymphocyte activation
.
Immunity
2011
;
35
:
871
82
.
32.
Heinzel
S
,
Binh Giang
T
,
Kan
A
,
Marchingo
JM
,
Lye
BK
,
Corcoran
LM
, et al
A Myc-dependent division timer complements a cell-death timer to regulate T cell and B cell responses
.
Nat Immunol
2017
;
18
:
96
103
.
33.
DeGregori
J
. 
E2F and cell survival: context really is key
.
Dev Cell
2005
;
9
:
442
4
.
34.
Preston
GC
,
Sinclair
LV
,
Kaskar
A
,
Hukelmann
JL
,
Navarro
MN
,
Ferrero
I
, et al
Single cell tuning of Myc expression by antigen receptor signal strength and interleukin-2 in T lymphocytes
.
EMBO J
2015
;
34
:
2008
24
.
35.
Sinclair
LV
,
Rolf
J
,
Emslie
E
,
Shi
YB
,
Taylor
PM
,
Cantrell
DA
. 
Control of amino-acid transport by antigen receptors coordinates the metabolic reprogramming essential for T cell differentiation
.
Nat Immunol
2013
;
14
:
500
8
.
36.
Velica
P
,
Zech
M
,
Henson
S
,
Holler
A
,
Manzo
T
,
Pike
R
, et al
Genetic Regulation of Fate Decisions in Therapeutic T Cells to Enhance Tumor Protection and Memory Formation
.
Cancer Res
2015
;
75
:
2641
52
.
37.
Denizot
F
,
Brunet
JF
,
Roustan
P
,
Harper
K
,
Suzan
M
,
Luciani
MF
, et al
Novel structures CTLA-2 alpha and CTLA-2 beta expressed in mouse activated T cells and mast cells and homologous to cysteine proteinase proregions
.
Eur J Immunol
1989
;
19
:
631
5
.
38.
Garcia-Bernal
D
,
Dios-Esponera
A
,
Sotillo-Mallo
E
,
Garcia-Verdugo
R
,
Arellano-Sanchez
N
,
Teixido
J
. 
RGS10 restricts upregulation by chemokines of T cell adhesion mediated by alpha4beta1 and alphaLbeta2 integrins
.
J Immunol
2011
;
187
:
1264
72
.
39.
Wu
H
,
Liu
G
,
Li
C
,
Zhao
S
. 
bri3, a novel gene, participates in tumor necrosis factor-alpha-induced cell death
.
Biochem Biophys Res Commun
2003
;
311
:
518
24
.
40.
Collins
S
,
Waickman
A
,
Basson
A
,
Kupfer
A
,
Licht
JD
,
Horton
MR
, et al
Regulation of CD4(+) and CD8(+) effector responses by Sprouty-1
.
PLoS One
2012
;
7
:
e49801
.
41.
Omilusik
KD
,
Shaw
LA
,
Goldrath
AW
. 
Remembering one's ID/E-ntity: E/ID protein regulation of T cell memory
.
Curr Opin Immunol
2013
;
25
:
660
6
.
42.
Zhou
X
,
Xue
HH
. 
Cutting edge: generation of memory precursors and functional memory CD8+ T cells depends on T cell factor-1 and lymphoid enhancer-binding factor-1
.
J Immunol
2012
;
189
:
2722
6
.
43.
Bronk
CC
,
Yoder
S
,
Hopewell
EL
,
Yang
S
,
Celis
E
,
Yu
XZ
, et al
NF-kappaB is crucial in proximal T-cell signaling for calcium influx and NFAT activation
.
Eur J Immunol
2014
;
44
:
3741
6
.
44.
Giordano
M
,
Roncagalli
R
,
Bourdely
P
,
Chasson
L
,
Buferne
M
,
Yamasaki
S
, et al
The tumor necrosis factor alpha-induced protein 3 (TNFAIP3, A20) imposes a brake on antitumor activity of CD8 T cells
.
Proc Natl Acad Sci U S A
2014
;
111
:
11115
20
.
45.
Zhong
G
,
Cheng
X
,
Long
H
,
He
L
,
Qi
W
,
Xiang
T
, et al
Dynamically expressed microRNA-15b modulates the activities of CD8+ T lymphocytes in mice with Lewis lung carcinoma
.
J Transl Med
2013
;
11
:
71
.
46.
Tegla
CA
,
Cudrici
CD
,
Nguyen
V
,
Danoff
J
,
Kruszewski
AM
,
Boodhoo
D
, et al
RGC-32 is a novel regulator of the T-lymphocyte cell cycle
.
Exp Mol Pathol
2015
;
98
:
328
37
.
47.
Topalian
SL
,
Drake
CG
,
Pardoll
DM
. 
Immune checkpoint blockade: a common denominator approach to cancer therapy
.
Cancer Cell
2015
;
27
:
450
61
.
48.
Spranger
S
,
Luke
JJ
,
Bao
R
,
Zha
Y
,
Hernandez
KM
,
Li
Y
, et al
Density of immunogenic antigens does not explain the presence or absence of the T-cell-inflamed tumor microenvironment in melanoma
.
Proc Natl Acad Sci U S A
2016
;
113
:
E7759
68
.
49.
Croft
M
. 
The role of TNF superfamily members in T-cell function and diseases
.
Nat Rev Immunol
2009
;
9
:
271
85
.
50.
Wherry
EJ
,
Kurachi
M
. 
Molecular and cellular insights into T cell exhaustion
.
Nat Rev Immunol
2015
;
15
:
486
99
.
51.
Kamphorst
AO
,
Wieland
A
,
Nasti
T
,
Yang
S
,
Zhang
R
,
Barber
DL
, et al
Rescue of exhausted CD8 T cells by PD-1-targeted therapies is CD28-dependent
.
Science
2017
;
355
:
1423
7
.
52.
West
EE
,
Jin
HT
,
Rasheed
AU
,
Penaloza-Macmaster
P
,
Ha
SJ
,
Tan
WG
, et al
PD-L1 blockade synergizes with IL-2 therapy in reinvigorating exhausted T cells
.
J Clinical Invest
2013
;
123
:
2604
15
.
53.
Huang
AC
,
Postow
MA
,
Orlowski
RJ
,
Mick
R
,
Bengsch
B
,
Manne
S
, et al
T-cell invigoration to tumour burden ratio associated with anti-PD-1 response
.
Nature
2017
;
545
:
60
5
.