The success of nanoparticle-based cancer therapeutics relies on their efficient tumor uptake and retention. Given this, improving nanoparticle localization in tumors is paramount to maximize their therapeutic potential. A common approach to achieve this is to functionalize nanoparticles with active targeting moieties that bind to specific tumor-associated receptors. Among these, arginine-glycine-aspartic acid (RGD) peptides have shown a potential to promote tumor accumulation by targeting the ανβ3 integrin receptor, a receptor commonly overexpressed by tumors owing to its role in promoting angiogenesis, metastasis and proliferation. Yet, its efficacy is commonly assessed using immunocompromised mice models. While useful, these models do not accurately account for immune-related interactions, which could lead to an overestimation of targeting efficacy. In our study, we investigated the efficacy of RGD peptides to improve the tumor accumulation of PEGylated gold nanoparticles (GNPs) using an immunocompetent mouse model. While RGD functionalization increased GNP uptake in cancer cells in vitro, it significantly reduced tumor accumulation in vivo due to enhanced off-target clearance by the mononuclear phagocyte system, with elevated accumulation in the spleen and liver. These findings highlight that RGD functionalization can promote immune-driven clearance in vivo, despite improving GNP uptake in cancer cells in vitro, emphasizing the importance of assessing targeting strategies in immunocompetent models for more physiologically relevant assessments.

Representative immune profiling flow plots and splenic and tumor immune profiling.

Homeostatic immunoregulatory mechanisms that prevent adverse effects of immune overaction can serve as barriers to successful anticancer immunity, representing attractive targets to improve cancer immunotherapy. Here, we demonstrated the role of the nonreceptor tyrosine kinase Fes, abundantly expressed in immune cells, as an innate intracellular immune checkpoint. Host Fes deficiency delayed tumor onset in a gene dose-dependent manner and improved tumor control, survival, doxorubicin efficacy, and sensitized tumors to anti-PD-1 therapy in murine triple-negative breast cancer and melanoma models. These effects were associated with a shift to an antitumorigenic immune microenvironment. Fes-deficient macrophages displayed increased Toll-like receptor signaling, proinflammatory cytokine production, and antigen presentation to and activation of T cells, leading to increased cancer cell killing in vitro and tumor control in vivo. This study highlights Fes as an innate immune checkpoint with potential as a therapeutic target and a predictive biomarker to guide immune checkpoint inhibitor treatment.Significance: Fes activity modulates the inflammatory cytokine presentation and T-cell priming capabilities of macrophages, supporting the potential of Fes as a target for developing therapeutic and biomarker strategies to improve cancer immunotherapy.

Immunotherapy is a powerful pillar of cancer therapy that still faces barriers due to cancer-mediated immunosuppression. Immunogenic cell death recruits and activates antigen-presenting cells (APCs) through engagement of pattern recognition receptors (PRRs), leading to pro-inflammatory Signal 3 cytokine production required for full activation of adaptive immune cells, including cytotoxic T lymphocytes (CTLs). The Fes tyrosine kinase suppresses innate immune responses in APCs by inhibiting PRR signaling and regulating cytokine trafficking. In non-cancer contexts, Fes may guard against consequences of overactive innate immunity, such as endotoxic shock or autoimmunity. However, this inhibitory effect on APCs may serve as a checkpoint to suppress successful anti-cancer immunotherapy by obstructing efficient priming of cancer specific CTLs. We hypothesize that Fes inhibition in APCs will improve Signal 3 cytokine signaling, resulting in greater adaptive immunity and tumor control. Using bone marrow derived macrophages (BMDMs) from wildtype (WT) or Fes knockout (fes-/-) mice, we assessed LPS-induced PRR signalling by immunoblot analysis, production of key Signal 3 cytokines by multiplex ELISA, and the level of cell-associated cytokines by flow cytometry. The ability of BMDMs to prime CTLs was assessed in antigen cross-presentation co-cultures with OT-1 T cells. Using E0771 and B16F10 syngeneic orthotopic mouse engraftment models, we compared the efficacy of doxorubicin and anti-PD-1 to control tumor growth and prolong survival in WT and fes-/- mice. Tumors and spleen immune profiles were analyzed by flow cytometry. WT or fes-/- BMDMs were adoptively transferred into E0771 and B16F10 tumours to directly assess their relative ability to control tumour growth. fes-/- BMDMs displayed improved PRR signaling, increased IFNβ, IL-12, and TNFα secretion, and greater levels of internal and cell-surface IL-12 following LPS stimulation. In antigen cross-presentation assays, LPS stimulated fes-/- BMDMs had improved CTL priming. Tumor control and survival were greater in fes-/- mice in both the E0771 and B16F10 engraftment models. Doxorubicin and the combination of doxorubicin and anti-PD-1 were more effective in fes-/-. This was especially striking in the B16F10 model, which is known to be resistant to PD-1 therapy. Immune profiling of tumors showed increased CTL and NK cell activation and PD-1 positivity, and a shift from predominately M2- to M1-polarized tumor associated macrophages in fes-/- mice. Intratumorally injected M1-polarized fes-/- BMDMs were better able to control tumor growth in both E0771 and B16F10 models. Fes acts as an innate immune checkpoint whose inhibition may enhance anti-cancer immunotherapy. We believe this effect is due to an increase in inflammatory signalling, and improved signal 3 cytokine signalling in fes-/- APCs. Brian J. Laight, Danielle Harper, Natasha Dmytryk, Connie S. Zhang, Changnian Shi, Sameh Basta, Madhuri Koti, Peter A. Greer. The Fes kinase plays an innate immune checkpoint role to suppress CD8+ T cell mediated anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6132.

Abstract Immunotherapies are a promising emerging pillar of cancer treatment, but they still face many barriers due to the immunosuppressive nature of cancer. Cancer immunotherapy relies on the interplay between innate and adaptive immune responses. One way of stimulating such responses, known as immunogenic cell death (ICD), involves the release of tumour-associated antigens and damage associated molecular patterns (DAMPs). These DAMPs function to recruit and activate innate immune cells, including antigen-presenting cells (APCs), through engagement of pattern recognition receptors (PRRs), subsequently leading to production of the pro-inflammatory Signal 3 cytokines required for activation of adaptive immune cells (e.g., cytotoxic T lymphocytes [CTLs] and natural killer [NK] cells). The tyrosine kinase Fes suppresses innate immune responses in APCs by inhibiting components of the PRR signaling cascade. In non-cancer contexts, the negative regulation of APCs by Fes may guard against consequences of overactive innate immunity, including endotoxic shock or autoimmune disease. However, this same inhibitory effect on APC function may also serve as a checkpoint to successful anti-cancer immunotherapy, by obstructing efficient priming of cancer specific CTLs by APCs. Therefore, by inhibiting Fes, we hypothesize there will be greater Signal 3 cytokine production, resulting in greater CTL activation, and therefore improved tumor control. Using bone marrow derived APCs, including macrophages (BMDMs) and dendritic cells (BMDCs), from wildtype (WT) or Fes knockout (fes-/-) mice, we have shown through both Western blotting and flow cytometry analysis, that PRR signal transduction cascades are suppressed by Fes and increase levels of Signal 3 cytokines produced by fes-/- APCs. This includes higher levels of cell associated IL-12 in fes-/- APCs. Using syngeneic orthotopic mouse engraftment models of triple negative breast cancer (EO771) and melanoma (B16-F10) we showed that treatment with doxorubicin (which induces ICD) or anti-PD-1 (immune checkpoint inhibitor) plus doxorubicin controls tumor growth and prolongs survival to a greater extent in fes-/- mice. Immunophenotyping of tumors and spleens from these mice showed higher levels of activated CTLs and skewing of macrophages to a M1 state in fes-/- mice. SIINFEKL peptide loaded-BMDM/BMDCs from fes-/- mice were also more effective at priming CTLs from OT-1 mice (which express a T cell receptor that recognizes the SIINFEKL peptide) in antigen cross-presentation co-culture assays. These results implicate Fes as a potential novel immune checkpoint whose inhibition may enhance anti-cancer immunotherapy by suppressing its role in dampening inflammatory Signal 3 cytokine production by APCs. I will present recent data exploring the role of Fes in regulating the expression and trafficking of IL-12 in APCs to better understand the molecular basis of improved CTL activation by fes-/- APCs. Citation Format: Julian Simonetti, Brian J. Laight, Natasha Dmytryk, Danielle Harper, Yan Gao, Changnian Shi, Madhuri Koti, Sameh Basta, Peter A. Greer. The FES tyrosine kinase as an emerging target for cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Optimizing Therapeutic Efficacy and Tolerability through Cancer Chemistry; 2024 Dec 9-11; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(12_Suppl):Abstract nr A017.

Activating the immune system is crucial for successful cancer immunotherapies, various proteins, such as the Fes non-receptor tyrosine kinase exist to limit activation and maintain homeostasis. However, in cancer settings, this serves as a barrier to the desired effects of immune activation following immunomodulatory treatment. Here, we demonstrate the role of Fes, a protein abundantly expressed in macrophages, as a novel innate intracellular immune checkpoint. Fes inactivity is associated with delayed tumour onset in a dose-dependent manner, and its deletion delays tumour growth, improves survival, enhances response to doxorubicin treatment, and sensitizes resistant tumours to PD-1 immune checkpoint inhibition. These effects are associated with an increase in Toll-like receptor signaling in antigen presenting cells, leading to an increase in proinflammatory cytokine production and cytotoxic T cell effector functions. Furthermore, we demonstrate a novel role for Fes in regulating the presentation of IL-12 on macrophage cell surfaces to enhance T-cell activation. Our results highlight Fes as a novel innate immune checkpoint with potential to serve as predictive biomarker to effective immune checkpoint blockade, and a potential novel therapeutic target for improved response to immunotherapy.### Competing Interest StatementThe authors have declared no competing interest.

[This corrects the article DOI: 10.3389/fimmu.2017.01629.].

Background Macrophages (M φ ) can exist along a spectrum of phenotypes that include pro-inflammatory (M1) or anti-inflammatory (M2) immune cells. M φ colony stimulating factor (M-CSF) and granulocyte M φ colony stimulating factor (GM-CSF) are cytokines important in hematopoiesis, polarization and activation of M φ . Methods and results To gain a greater understanding of the relationship between GM-CSF and M-CSF, we investigated an in vitro model of differentiation to determine if GM-CSF and M-CSF can antagonize each other, in terms of M φ phenotype and functions. We determined that M φ cultured in mixed M-CSF: GM-CSF ratios exhibit M1-like GM-CSF-treated macrophage phenotype when the ratios of the two cytokines are 1:1 in culture. Moreover, GM-CSF is dominant over M-CSF in influencing M φ production of proinflammatory cytokines such as IL-6, TNFα, and IL-12p40, and the anti-inflammatory cytokine IL-10. Conclusions Our data established that GM-CSF is more dominant over M-CSF, triggering the M φ to become pro-inflammatory cells. These findings provide insight into how GM-CSF can influence M φ activation with implications in inflammatory diseases where the M φ status can play a significant role in supporting the inflammatory conditions.

Macrophages (Mφ) are innate immune cells with a variety of functional phenotypes depending on the cytokine microenvironment they reside in. Mφ exhibit distinct activation patterns that are found within a wide array of activation states ranging from the originally discovered classical pro-inflammatory (M1) to the anti-inflammatory (M2) with their multi-facades. M1 cells are induced by IFNγ + LPS, while M2 are further subdivided into M2a (IL-4), M2b (Immune Complex) and M2c (IL-10) based on their inducing stimuli. Not surprisingly, Mφ activation influences the outcome of viral infections as they produce cytokines that in turn activate cells of the adaptive immune system. Generally, activated M1 cells tend to restrict viral replication, however, influenza and HIV exploit inflammation to support their replication. Moreover, M2a polarization inhibits HIV replication at the post-integration level, while HCMV encoded hrIL-10 suppresses inflammatory reactions by facilitating M2c formation. Additionally, viruses such as LCMV and Lassa Virus directly suppress Mφ activation leading to viral chronicity. Here we review how Mφ activation affects viral infection and the strategies by which viruses manipulate Mφ polarization to benefit their own fitness. An understanding of these mechanisms is important for the development of novel immunotherapies that can sway Mφ phenotype to inhibit viral replication.

The protocol used to induce cell death for generating vaccines from whole tumor cells is a critical consideration that impacts vaccine efficacy. Here we compared how different protocols used to induce cell death impacted protection provided by a prophylactic whole tumor cell vaccine in a mouse melanoma model. We found that melanoma cells exposed to γ-irradiation or lysis combined with UV-irradiation (LyUV) provided better protection against tumor challenge than lysis only or cells exposed to UV-irradiation. Furthermore, we found that the immunoregulatory cytokine, IL-27 enhanced protection against tumor growth in a dose-dependent manner when combined with either LyUV or γ-irradiated whole tumor cell vaccine preparations. Taken together, this data supports the use of LyUV as a potential protocol for developing whole tumor cell prophylactic cancer vaccines. We also showed that IL-27 can be used at low doses as a potent adjuvant in combination with LyUV or γ-irradiation treated cancer cells to improve the protection provided by a prophylactic cancer vaccine in a mouse melanoma model.

GM-CSF acts as a pro-inflammatory cytokine and a key growth factor produced by several immune cells such as macrophages and activated T cells. In this review, we discuss recent studies that point to the crucial role of GM-CSF in the immune response against infections. Upon induction, GM-CSF activates four main signalling networks including the JAK/STAT, PI3K, MAPK, and NFκB pathways. Many of these transduction pathways such as JAK/STAT signal via proteins commonly activated with other antiviral signalling cascades, such as those induced by IFNs. GM-CSF also helps defend against respiratory infections by regulating alveolar macrophage differentiation and enhancing innate immunity in the lungs. Here, we also summarize the numerous clinical trials that have taken advantage of GM-CSF's mechanistic attributes in immunotherapy. Moreover, we discuss how GM-CSF is used as an adjuvant in vaccines and how its activity is interfered with to reduce inflammation such as in the case of COVID-19. This review brings forth the current knowledge on the antiviral actions of GM-CSF, the associated signalling cascades, and its application in immunotherapy.

Regulation of proinflammatory cytokine expression is critical in the face of single-stranded RNA (ssRNA) virus infections. Many viruses, including coronavirus and influenza virus, wreak havoc on the control of cytokine expression, leading to the formation of detrimental cytokine storms. Understanding the regulation and interplay between inflammatory cytokines is critical to the identification of targets involved in controlling the induction of cytokine expression. In this study, we focused on how the antiviral cytokine interleukin-27 (IL-27) regulates signal transduction downstream of Toll-like receptor 7 (TLR7) and TLR8 ligation, which recognize endosomal single-stranded RNA. Given that IL-27 alters bacterial-sensing TLR expression on myeloid cells and can inhibit replication of single-stranded RNA viruses, we investigated whether IL-27 affects expression and function of TLR7 and TLR8. Analysis of IL-27-treated THP-1 monocytic cells and THP-1-derived macrophages revealed changes in mRNA and protein expression of TLR7 and TLR8. Although treatment with IL-27 enhanced TLR7 expression, only TLR8-mediated cytokine secretion was amplified. Furthermore, we demonstrated that imiquimod, a TLR7 agonist, inhibited cytokine and chemokine production induced by a TLR8 agonist, TL8-506. Delineating the immunomodulatory role of IL-27 on TLR7 and TLR8 responses provides insight into how myeloid cell TLR-mediated responses are regulated during virus infection.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) play an important role in macrophage (MФ) development by influencing their differentiation and polarization. Our goal was to explore the difference between M-CSF- and GM-CSF-derived bone marrow MФ responsiveness to TLR7-mediated signalling pathways that influence cytokine production early after infection in a model of acute virus infection. To do so, we examined cytokine production and TLR7-mediated signalling at 1 h post-lymphocytic choriomeningitis virus (LCMV) Armstrong (ARM) infection. We found that R848-induced cytokine expression was enhanced in these cells, with GM-CSF cells exhibiting higher proinflammatory cytokine expression and M-CSF cells exhibiting higher anti-inflammatory cytokine expression. However, R848-mediated signalling molecule activation was diminished in LCMV-infected M-CSF and GM-CSF macrophages. Interestingly, we observed that TLR7 expression was maintained during LCMV infection of M-CSF and GM-CSF cells. Moreover, TLR7 expression was significantly higher in M-CSF cells compared to GM-CSF cells. Taken together, our data demonstrate that although LCMV restrains early TLR7-mediated signalling, it primes differentiated MФ to enhance expression of their respective cytokine profiles and maintains levels of TLR7 expression early after infection.

Macrophages (Mϕ) are highly plastic, and can acquire a variety of functional phenotypes depending on the presence of different stimuli in their local environment. Mφ stimulated by interleukin (IL)-4 induce an alternative activation state and function as anti-inflammatory cells and promote tissue repair. However, there is overwhelming evidence that IL-4 can play a role in promoting inflammation. In asthma and allergic inflammation, IL-4 mediates proinflammatory responses that lead to tissue damage. Thus the effect of IL-4 on the outcome of the immune responses is greatly influenced by other cofactors and cytokines present in the microenvironment. R848 (resiquimod), a TLR7/8 agonist is a novel vaccine adjuvant, triggering a strong Th1-skewed response but its efficacy as a vaccine adjuvant shows variable results. It is not currently known whether the presence of IL-4 can dampen or enhance immunity in response to TLR7 agonists. In the present study, we sought to investigate the impact of IL-4-induced Mφ polarization on the outcome of R848 stimulation. The activation marker expression and production of cytokines were measured in murine spleen-derived Mφ. Protein expression levels of innate recognition molecules and transcription factors involved, including retinoic-acid inducible gene I, mitochondrial antiviral signaling protein, stimulator of interferon genes (STING), and IFN regulatory factors were evaluated in activated Mφ. These play a crucial role in the control of viral replication and optimal CD8+ T cell priming. We report that sustained priming with IL-4 alone promotes an antiviral response in Mφ, and enhances proinflammatory responses to R848 treatment. This highlights the need for better understanding of IL-4 proinflammatory functions and its potential use as a broad-acting antiviral in combination with R848 may be used in combination with other therapies to target the innate arm of immunity against emerging infections.

Granulocyte/macrophage colony-stimulating factor (GM-CSF) and macrophage CSF (M-CSF) modulate differentiation and immune functions of macrophages (MΦ). Our aim was to evaluate how different MΦ differentiation conditions influence the MΦ response to virus infection. To address this, we differentiated bone marrow-derived MΦ in either GM-CSF or M-CSF and measured the cytokine responses to two different strains of lymphocytic choriomeningitis virus (LCMV) (clone 13; Cl13 or Armstrong; ARM). GM-CSF MΦ infected with either LCMV-ARM or -Cl13 produced more IL-6 than M-CSF MΦ, whereas M-CSF MΦ generated more IL-10 than GM-CSF MΦ. Interestingly, in M-CSF MΦ, LCMV-ARM induced more IL-10 production than Cl13. However, we could not detect any IL-12p70 or IL-23 after infection from either cell types. We also observed that GM-CSF MΦ was more efficient than M-CSF MΦ in supporting antigen-specific CD8+ T cell proliferation. Taken together, our data demonstrate that GM-CSF and M-CSF MΦ differ in how they respond to viral infection by their production of different cytokines, and their support for CD8+ T cell proliferation.

The role of the immune system in anti-tumor immunity cannot be overstated, as it holds the potential to promote tumor eradication or prevent tumor cell escape. Cytokines are critical to influencing the immune responses and interactions with non-immune cells. Recently, the IL-12 and IL-6 family of cytokines have accumulated newly defined members each with specific immune functions related to various cancers and tumorigenesis. There is a need to better understand how cytokines like IL-27, IL-30, and IL-35 interact with one another, and how a developing tumor can exploit these interactions to enhance immune suppression. Current cytokine-based immunotherapies are associated with cytotoxic side effects which limits the success of treatment. In addition to this toxicity, understanding the complex interactions between immune and cancer cells may be one of the greatest challenges to developing a successful immunotherapy. In this review, we bring forth IL-27, IL-30, and IL-35, “sister cytokines,” along with more recent additions to the IL-12 family, which serve distinct purposes despite sharing structural similarities. We highlight how these cytokines function in the tumor microenvironment by examining their direct effects on cancer cells as well their indirect actions via regulatory functions of immune cells that act to either instigate or inhibit tumor progression. Understanding the context dependent immunomodulatory outcomes of these sister cytokines, as well as their regulation within the tumor microenvironment, may shed light onto novel cancer therapeutic treatments or targets.

Macrophages make up a crucial aspect of the immune system, carrying out a variety of functions ranging from clearing cellular debris to their well-recognized roles as innate immune cells. These cells exist along a spectrum of phenotypes but can be generally divided into proinflammatory (M1) and anti-inflammatory (M2) groups, representing different states of polarization. Due to their diverse functions, macrophages are implicated in a variety of diseases such as atherosclerosis, lupus nephritis, or infection with HIV. Throughout their lifetime, macrophages can be influenced by a wide variety of signals that influence their polarization states, which can affect their function and influence their effects on disease progression. This review seeks to provide a summary of how GM-CSF and M-CSF influence macrophage activity during disease, and provide examples of in vitro research that indicate competition between the two cytokines in governing macrophage polarization. Gaining a greater understanding of the relationship between GM-CSF and M-CSF, along with how these cytokines fit into the larger context of diseases, will inform their use as treatments or targets for treatment in various diseases.

Nearly a decade ago, an endoplasmic reticulum (ER) adaptor protein called stimulator of interferon genes (STING) was found to be critical in the induction of type I IFN production in response to DNA virus infection. STING functions by sensing cytoplasmic DNA and activates key transcription factors, including IFN regulatory factor (IRF)-3 and IRF7, to initiate type I IFN expression. Type I IFNs are vital in immunity against viral infections and can influence cancer cell proliferation, migration, and apoptosis. Several studies have shown that STING activation results in potent antitumor activity by generating strong tumor-specific cytotoxic T-cell responses. Moreover, compared with wild-type, STING-knockout mice show greater susceptibility to viral infections. In this review, we discuss the importance of STING signaling during the induction of immune responses, especially those associated with type I IFN in viral infections and tumor immunity. Furthermore, we highlight recent data that unravel how the STING signaling pathway can be negatively regulated.