CD137 (4-1BB) stimulation leads to metabolic and functional reprogramming of human monocytes/macrophages enhancing their tumoricidal activity

Immunotherapies have heralded a new era in the cancer treatment. In addition to checkpoint inhibitors, agonistic antibodies against co-stimulatory immune receptors hold the potential to invoke efficient antitumor immunity. Targeting CD137 has gained momentum based on its ability to drive NK- and T-cell-based responses. CD137-engaging mAbs have already entered clinical trials for different types of tumors showing promising results. Despite the efforts to translate CD137-mediated immunotherapy into clinical practice, little remains known regarding the role of CD137 in human monocytes/macrophages. We found CD137 being expressed on monocytes of healthy controls and at even higher levels in patients with multiple myeloma or CLL. CD137HI(GH) monocytes displayed a distinct phenotypic, transcriptomic, and metabolic profile. They possessed an increased phagocytic capacity enabling superior antibody-dependent phagocytosis (ADPC) of multiple myeloma and lymphoma cells that were treated with anti-CD38 or anti-CD20 mAbs. Triggering CD137 promoted both metabolic and tumoricidal activity in an extracellular signal-regulated kinase (ERK)-dependent fashion. In addition, we observed a phenotypic, transcriptomic, and functional skewing towards a M1-like phenotype. Overall, we introduce CD137 as a positive immune checkpoint on human monocytes/macrophages, which can have therapeutic implications especially in view of synergistic effects when combining CD137 agonists with tumor-targeting antibodies.

2 (Miltenyi Biotec) or human immunoglobulins (Grifols, Gamunex®) prior to the staining. Intracellular staining was performed with a Fixation/Permeabilization Solution Kit (BD Biosciences). For intracellular detection of phosphorylated proteins Phosflow TM Fix Buffer I and Phosflow TM Perm Buffer III (BD Biosciences) were used.
Glucose uptake was analyzed using the fluorescent glucose analog 6-NBDG (ThermoFisher Scientific). Fatty acid uptake was semi-quantified by use of the fluorescently labeled long chain fatty acid, BODIPY™ FL C16 (ThermoFisher Scientific). Mitochondrial superoxide production was assessed by MitoSOX™ Red (Thermo Fisher Scientific) staining according to the manufacturer's recommendations. To analyze the mitochondrial biomass and fitness Mito-Tracker® Green (ThermoFisher Scientific) and Tetramethylrhodamine ethyl ester (TMRE, Cayman Chemicals) were used respectively.

ADP/ATP Assay
ADP/ATP ratio of in vitro stimulated monocytes was assessed applying the ADP/ATP Ratio Assay Kit (Sigma-Aldrich) with 10 4 cells/ sample as per manufacturer's instructions.

Glucose and lactic acid
Glucose and lactic acid in cell culture supernatants were measured with a HITADO SuperGL compact system according to the manufacturer's instructions.

T-cell proliferation assay
In vitro stimulated monocytes were subsequently co-cultured with autologous VPD-labeled Tcells in different ratios. T-cells were stimulated for five days using anti-CD2, -CD3 and -CD28 bead-coupled antibodies (Miltenyi Biotec), and proliferation was assessed by FACS.
For detection of IL10-and IL12-producing cells after four days of CD137 stimulation, monocytes were harvested and reseeded at 0.4 x 10 6 /well in a 24 well plate. Next, cells were either activated with 1 µg/µl LPS and 2 µg/µl Resiquimod for 20 h or left untreated. Subsequently, two-color cytokine secretion assay was performed using IL-10 detection Kit (Miltenyi Biotec) and IL-12 detection Kit (Miltenyi Biotec).

DNA/RNA isolation, quantitative real-time PCR and RNA-sequencing
DNA and RNA were isolated from monocytes using the innuPREP DNA/RNA Mini Kit (analytic Jena). DNA/RNA content was quantified by means of a NanoDrop TM (Thermo Fisher Scientific). For qPCR, RNA was reverse transcribed into cDNA using the SuperScript™ II Reverse Transcriptase (Thermo Fisher Scientific). Gene expression was quantified using the Rotor-Gene SYBR® Green PCR Kit (Qiagen, 204076) and dedicated primers on a Rotor-Gene Q (Qiagen) employing the 2-standard method.

Stoll et al.
CD137 signaling promotes tumoricidal monocytes/macrophages 4 Samples designated for RNA-sequencing (RNAseq) were further processed for removal of residual genomic DNA using the RNase free DNase set (Qiagen).
For RNAseq of ex vivo sorted monocytes "INVIEW Transcriptome" (Eurofins/GATC Biotech) product was purchased and RNAseq was performed by Eurofins/GATC Biotech. A strandspecific cDNA library was prepared and sequencing was performed on a HiSeq4000 with >30 x 10 6 50bp single end reads.
RNA-sequencing of in vitro stimulated monocytes was performed by the next generation sequencing core unit of the FAU Erlangen-Nürnberg. The cDNA library was prepared using TruSeq Stranded mRNA Kit (Illumina). Libraries were sequenced on a HiSeq2500 sequencer (Illumina) with 95bp single end reads.