30 juni 2026
Lunger J.C. et al. BioRxiv
Keywords
Macrophage polarization
Synthetic immunology
CAR-macrophages
Main Findings
Macrophages are highly plastic immune cells that perform diverse functions, from fighting infections to maintaining tissue homeostasis. Their dysregulation contributes to a range of diseases, while their plasticity makes them attractive for cell therapy. The central challenge is to guide macrophage polarization to defined states precisely and stably with current approaches allowing only coarse control with limited stability. Since cytokines naturally direct macrophage polarization through receptor signalling, Lunger et al. developed synthetic cytokine receptors (SCRs) that activate signalling in a cytokine-independent manner via short linear signalling motifs from the intracellular domains of natural cytokine receptors.
The authors built SCRs containing motifs from nine cytokine receptors. SCRs carrying a motif from a single receptor induced surface marker expression and phagocytic activity comparable to the corresponding cytokine. For SCRs modelling anti-inflammatory (IL-10-based) and pro-inflammatory (IFNγ-based) signalling, deeper transcriptional analysis showed that their programs also resembled those of their cognate cytokines. Next, the authors generated a library of 131 SCRs containing up to three motifs from different receptors to determine if they could achieve macrophage polarization states beyond the classical M1/M2 paradigm. This indeed induced macrophage polarization states distinct from those achieved by single cytokines. To test if these findings could be applied to improve therapeutic function of macrophages, the authors incorporated the most phagocytic SCR from the library into CAR-macrophages. The resulting macrophages had enhanced in vitro killing of target cells compared to CAR-macrophages with an empty SCR and improved control of tumour burden in a murine xenograft model of human ovarian cancer.
Because increasing the number of motifs per SCR or including motifs from other cytokine receptors makes the combinatorial space too large to test experimentally, the authors built a two-state model trained on the SCR library data to predict how SCR composition guides macrophage polarization. The model was highly predictive and was used to forecast phenotypes for SCRs containing up to four motifs. Finally, the authors used the model to engineer an SCR inducing simultaneously pro-inflammatory and highly phagocytic macrophages, a desirable phenotype for cancer immunotherapy which was not observed in the previously prepared SCR library. Combining IFNγ- and IL-10-receptor motifs at a 1:3 ratio induced this state, and the corresponding SCR-CAR-macrophages killed target cells in vitro and secreted pro-inflammatory factors associated with cytotoxic T cell recruitment. Together, this work couples a modular receptor platform with a predictive model, enabling rational design of macrophage polarization states.
Limitations
The authors establish synthetic cytokine receptors as a valuable tool to rationally program macrophage polarization, supported by a strong experimental design with appropriate controls. However, we believe that the manuscript would benefit from these considerations:
The claim that SCRs drive stronger phenotypes than the corresponding cytokines is confounded by exposure time: cells were transduced with SCR on day 3 of a 9-day culture, whereas cytokines were added only 48 h before readout, on day 7.
Constitutive signalling of SCRs raises toxicity and metabolic exhaustion concerns. The effect of SCR signalling on macrophage viability and phenotypic as well as functional stability should be investigated.
It is unclear whether the improved killing by phagocytic SCR-CAR-Ms (SCR: S3-S3) is driven by phagocytosis. A phagocytosis-blocking control (e.g., cytoskeletal inhibition) would help establish whether phagocytosis is responsible for enhanced killing.
The intraperitoneal SKOV3 xenograft model with intraperitoneal administration of SCR-CAR-macrophages primarily tests local killing rather than trafficking. A systemic intravenous administration would assess macrophage homing.
NSG mice lack a functional adaptive immune system, yet the rationale for the pro-inflammatory CXCL9/CXCL10 program is cytotoxic T cell recruitment. A humanized or otherwise T cell–competent model with a readout of intratumoral T cell infiltration is recommended.
The model-designed SCR(S3-S3-S3-S1) killed target cancer cells to a similar level as the simpler library-guided SCR(S3-S3) in vitro but has not been tested in vivo, so the rational-design achievement currently lacks a demonstrated functional benefit. A comparison in vivo would be recommended.
The main text of the preprint is very well written with logical information flow, largely in sync with the main figures. However, several extended data figures (Extended Data Figures 5 and 6 and several panels of Extended Data Figure 3) were not mentioned in the text. We recommend ensuring all figures and extended data figures are cited at the relevant points in the text.
Significance/Novelty
This study introduces synthetic cytokine receptors for precise programming of macrophage polarization state, replacing the coarse control via exogenous cytokines with constitutive, motif-based signalling, potentially making the polarization state less dependent on environmental cues. Furthermore, SCRs will allow for studying how macrophages integrate multiple signalling inputs to determine their phenotype and function. The authors showed that this signal integration could be modelled with a simple two-state model predicting phenotype from motif combination accurately enough to engineer novel receptors that escape known limitations, such as a trade-off between phagocytosis and inflammatory signalling. We expect this work to be of broad interest because the rational engineering of macrophage polarization has great potential for improving cellular immunotherapies, from anti-tumour CAR-Ms to regulatory and regenerative macrophages.
Credit
Reviewed by Tomislav Kostevc as part of a cross-institutional journal club between the Max-Delbrück Center Berlin, the Ragon Institute Boston (Mass General, MIT, Harvard), the University of Virginia, the Medical University of Vienna and other life science institutes in Vienna.
The author declares no conflict of interests in relation to their involvement in the review.