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The combination of radiotherapy (RT) with immunotherapy represents a promising treatment modality for non-small cell lung cancer (NSCLC) patients. As only a minority of patients shows a persistent response today, a spacious optimization window remains to be explored. Previously we showed that fractionated RT can induce a local immunosuppressive profile. Based on the evolving concept of an immunomodulatory role for vagal nerve stimulation (VNS), we tested its therapeutic and immunological effects alone and in combination with fractionated RT in a preclinical-translational study. Lewis lung carcinoma-bearing C57Bl/6 mice were treated with VNS, fractionated RT or the combination while a patient cohort with locally advanced NSCLC receiving concurrent radiochemotherapy (ccRTCT) was enrolled in a clinical trial to receive either sham or effective VNS daily during their 6 weeks of ccRTCT treatment. Preclinically, VNS alone or with RT showed no therapeutic effect yet VNS alone significantly enhanced the activation profile of intratumoral CD8 + T cells by upregulating their IFN-γ and CD137 expression. In the periphery, VNS reduced the RT-mediated rise of splenic, but not blood-derived, regulatory T cells (Treg) and monocytes. In accordance, the serological levels of protumoral CXCL5 next to two Treg-attracting chemokines CCL1 and CCL22 were reduced upon VNS monotherapy. In line with our preclinical findings on the lack of immunological changes in blood circulating immune cells upon VNS, immune monitoring of the peripheral blood of VNS treated NSCLC patients (n=7) did not show any significant changes compared to ccRTCT alone. As our preclinical data do suggest that VNS intensifies the stimulatory profile of the tumor infiltrated CD8 + T cells, this favors further research into non-invasive VNS to optimize current response rates to RT-immunotherapy in lung cancer patients.

BackgroundPoorly immunogenic tumors are hardly responsive to immunotherapies such as immune checkpoint blockade (ICB) and are, therefore, a therapeutic challenge. Combination with other immunotherapies and/or immunogenic therapies, such as radiotherapy (RT), could make these tumors more immune responsive. We have previously shown that the immunocytokine L19–IL2 combined with single-dose RT resulted in 75% tumor remission and a 20% curative abscopal effect in the T cell-inflamed C51 colon carcinoma model. This treatment schedule was associated with the upregulation of inhibitory immune checkpoint (IC) molecules on tumor-infiltrating T cells, leading to only tumor growth delay in the poorly immunogenic Lewis lung carcinoma (LLC) model.MethodsWe aimed to trigger curative therapeutic responses in three tumor models (LLC, C51 and CT26) by “pushing the accelerator” of tumor immunity with L19–IL2 and/or “releasing the brakes” with ICB, such as antibodies directed against cytotoxic T lymphocyte associated protein 4 (CTLA-4), programmed death 1 (PD-1) or its ligand (PD-L1), combined with single-dose RT (10 Gy or 5 Gy). Primary tumor endpoint was defined as time to reach four times the size of tumor volume at start of treatment (4T×SV). Multivariate analysis of 4T×SV was performed using the Cox proportional hazards model comparing each treatment group with controls. Causal involvement of T and natural killer (NK) cells in the anti-tumor effect was assessed by in vivo depletion of T, NK or both cell populations. Immune profiling was performed using flow cytometry on single cell suspensions from spleens, bone marrow, tumors and blood.ResultsCombining RT, anti-PD-L1 and L19–IL2 cured 38% of LLC tumors, which was both CD8+ T and NK cell dependent. LLC tumors were resistant to RT +anti-PD-L1 likely explained by the upregulation of other IC molecules and increased T regulatory cell tumor infiltration. RT+L19–IL2 outperformed RT+ICB in C51 tumors; effects were comparable in CT26 tumors. Triple combinations were not superior to RT+L19–IL2 in both these models.ConclusionsThis study demonstrated that combinatorial strategies rationally designed on biological effects can turn immunotherapy-resistant tumors into immunologically responsive tumors. This hypothesis is currently being tested in the international multicentric randomized phase 2 trial: ImmunoSABR (NCT03705403).

Tumor extracellular matrix has been associated with drug resistance and immune suppression. Here, proteomic and RNA profiling reveal increased collagen levels in lung tumors resistant to PD-1/PD-L1 blockade. Additionally, elevated collagen correlates with decreased total CD8 + T cells and increased exhausted CD8 + T cell subpopulations in murine and human lung tumors. Collagen-induced T cell exhaustion occurs through the receptor LAIR1, which is upregulated following CD18 interaction with collagen, and induces T cell exhaustion through SHP-1. Reduction in tumor collagen deposition through LOXL2 suppression increases T cell infiltration, diminishes exhausted T cells, and abrogates resistance to anti-PD-L1. Abrogating LAIR1 immunosuppression through LAIR2 overexpression or SHP-1 inhibition sensitizes resistant lung tumors to anti-PD-1. Clinically, increased collagen, LAIR1, and TIM-3 expression in melanoma patients treated with PD-1 blockade predict poorer survival and response. Our study identifies collagen and LAIR1 as potential markers for immunotherapy resistance and validates multiple promising therapeutic combinations. Tumor extracellular matrix has been associated with cancer progression, therapy resistance and immune suppression. Here, the authors show that collagen generates resistance to PD-1/PD-L1 immunotherapy by upregulating LAIR1 expression and downstream signaling, leading to increased CD8+ T cell exhaustion.

BackgroundModulation and depletion strategies of regulatory T cells (Tregs) constitute valid approaches in antitumor immunotherapy but suffer from severe adverse effects due to their lack of selectivity for the tumor-infiltrating (ti-)Treg population, indicating the need for a ti-Treg specific biomarker.MethodsWe employed single-cell RNA-sequencing in a mouse model of non-small cell lung carcinoma (NSCLC) to obtain a comprehensive overview of the tumor-infiltrating T-cell compartment, with a focus on ti-Treg subpopulations. These findings were validated by flow cytometric analysis of both mouse (LLC-OVA, MC38 and B16-OVA) and human (NSCLC and melanoma) tumor samples. We generated two CCR8-specific nanobodies (Nbs) that recognize distinct epitopes on the CCR8 extracellular domain. These Nbs were formulated as tetravalent Nb-Fc fusion proteins for optimal CCR8 binding and blocking, containing either an antibody-dependent cell-mediated cytotoxicity (ADCC)-deficient or an ADCC-prone Fc region. The therapeutic use of these Nb-Fc fusion proteins was evaluated, either as monotherapy or as combination therapy with anti-programmed cell death protein-1 (anti-PD-1), in both the LLC-OVA and MC38 mouse models.ResultsWe were able to discern two ti-Treg populations, one of which is characterized by the unique expression of Ccr8 in conjunction with Treg activation markers. Ccr8 is also expressed by dysfunctional CD4+ and CD8+ T cells, but the CCR8 protein was only prominent on the highly activated and strongly T-cell suppressive ti-Treg subpopulation of mouse and human tumors, with no major CCR8-positivity found on peripheral Tregs. CCR8 expression resulted from TCR-mediated Treg triggering in an NF-κB-dependent fashion, but was not essential for the recruitment, activation nor suppressive capacity of these cells. While treatment of tumor-bearing mice with a blocking ADCC-deficient Nb-Fc did not influence tumor growth, ADCC-prone Nb-Fc elicited antitumor immunity and reduced tumor growth in synergy with anti-PD-1 therapy. Importantly, ADCC-prone Nb-Fc specifically depleted ti-Tregs in a natural killer (NK) cell-dependent fashion without affecting peripheral Tregs.ConclusionsCollectively, our findings highlight the efficacy and safety of targeting CCR8 for the depletion of tumor-promoting ti-Tregs in combination with anti-PD-1 therapy.

Only a subpopulation of non-small cell lung cancer (NSCLC) patients responds to immunotherapies, highlighting the urgent need to develop therapeutic strategies to improve patient outcome. We develop a chemical positive modulator (HEI3090) of the purinergic P2RX7 receptor that potentiates αPD-1 treatment to effectively control the growth of lung tumors in transplantable and oncogene-induced mouse models and triggers long lasting antitumor immune responses. Mechanistically, the molecule stimulates dendritic P2RX7-expressing cells to generate IL-18 which leads to the production of IFN-γ by Natural Killer and CD4 + T cells within tumors. Combined with immune checkpoint inhibitor, the molecule induces a complete tumor regression in 80% of LLC tumor-bearing mice. Cured mice are also protected against tumor re-challenge due to a CD8-dependent protective response. Hence, combination treatment of small-molecule P2RX7 activator followed by immune checkpoint inhibitor represents a strategy that may be active against NSCLC. A limited percentage of patients with non-small cell lung cancer respond to immunotherapy. Here the authors show that HEI3090, a chemical positive modulator of the purinergic P2RX7 receptor, promotes IL-18 mediated anti-tumor immune responses and sensitizes lung cancer to anti-PD-1 therapy in preclinical models.

Involvement of the immune system in tumour progression is at the forefront of cancer research. Analysis of the tumour immune microenvironment has yielded a wealth of information on tumour biology, and alterations in some immune subtypes, such as tumour-associated macrophages (TAM), can be strong prognostic indicators. Here, we use optical tissue clearing and a TAM-targeting injectable fluorescent nanoparticle (NP) to examine three-dimensional TAM composition, tumour-to-tumour heterogeneity, response to colony-stimulating factor 1 receptor (CSF-1R) blockade and nanoparticle-based drug delivery in murine pulmonary carcinoma. The method allows for rapid tumour volume assessment and spatial information on TAM infiltration at the cellular level in entire lungs. This method reveals that TAM density was heterogeneous across tumours in the same animal, overall TAM density is different among separate pulmonary tumour models, nanotherapeutic drug delivery correlated with TAM heterogeneity, and successful response to CSF-1R blockade is characterized by enhanced TAM penetration throughout and within tumours. Tumour-associated macrophages (TAM) can be used as prognostic indicators in cancer. Here, the authors establish a platform for high-throughput 3D microscopy in murine lung carcinoma that allows to visualize TAMs infiltration throughout the entire lung, response to CSF-1R blockade and nanoparticle drug delivery.

Neoantigen-specific T cells are strongly implicated as being critical for effective immune checkpoint blockade treatment (ICB) (e.g., anti–PD-1 and anti–CTLA-4) and are being targeted for vaccination-based therapies. However, ICB treatments show uneven responses between patients, and neoantigen vaccination efficiency has yet to be established. Here, we characterize neoantigen-specific CD8⁺ T cells in a tumor that is resistant to ICB and neoantigen vaccination. Leveraging the use of mass cytometry combined with multiplex major histocompatibility complex (MHC) class I tetramer staining, we screened and identified tumor neoantigen–specific CD8⁺ T cells in the Lewis Lung carcinoma (LLC) tumor model (mRiok1). We observed an expansion of mRiok1-specific CD8⁺ tumor-infiltrating lymphocytes (TILs) after ICB targeting PD-1 or CTLA-4 with no sign of tumor regression. The expanded neoantigen-specific CD8⁺ TILs remained phenotypically and functionally exhausted but displayed cytotoxic characteristics. When combining both ICB treatments, mRiok1-specific CD8⁺ TILs showed a stem-like phenotype and a higher capacity to produce cytokines, but tumors did not show signs of regression. Furthermore, combining both ICB treatments with neoantigen vaccination did not induce tumor regression either despite neoantigen-specific CD8⁺ TIL expansion. Overall, this work provides a model for studying neoantigens in an immunotherapy nonresponder model. We showed that a robust neoantigen-specific T-cell response in the LLC tumor model could fail in tumor response to ICB, which will have important implications in designing future immunotherapeutic strategies.

BackgroundMany anti-angiogenic agents have the potential to modulate the tumor microenvironment and improve immunotherapy. Anlotinib has demonstrated anti-tumor efficacy in non-small cell lung cancer (NSCLC) in third-line clinical trials. However, its roles in immune regulation and potentially synergistic anti-tumor effect in combination with immune checkpoint inhibition remain unclear.MethodsHere, based on a syngeneic lung cancer mouse model, the intratumoral immunological changes post-anlotinib treatment in the model were assessed. Furthermore, it was tested whether anlotinib could enhance the anti-tumor effect of αPD-1 in vivo.ResultsThis study shows that anlotinib increased infiltration of the innate immune cells, including natural killer (NK) cells, and antigen-presenting cells (APC), which include M1-like tumor-associated macrophages (TAM) and dendritic cells (DC), whereas the percentage of M2-like TAM was dramatically reduced. Subsequently, when combined with PD-1/PD-L1 (programmed cell death 1/PD-1 ligand 1) blockade, anlotinib conferred significantly synergistic therapeutic benefits.ConclusionsOverall, these findings describe a role for anlotinib in the innate immune cells in the tumor microenvironment and a potentially synergistic anti-tumor combination with immune checkpoint inhibition.

Treatment of tumors with ionizing radiation stimulates an antitumor immune response partly dependent on induction of IFNs. These IFNs directly enhance dendritic cell and CD8+ T cell activity. Here we show that resistance to an effective antitumor immune response is also a result of IFN signaling in a different cellular compartment of the tumor, the cancer cells themselves. We abolished type I IFN signaling in cancer cells by genetic elimination of its receptor, IFNAR1. Pronounced immune responses were provoked after ionizing radiation of tumors from 4 mouse cancer cell lines with Ifnar1 knockout. This enhanced response depended on CD8+ T cells and was mediated by enhanced susceptibility to T cell-mediated killing. Induction of Serpinb9 proved to be the mechanism underlying control of susceptibility to T cell killing after radiation. Ifnar1-deficient tumors had an augmented response to anti-PD-L1 immunotherapy with or without radiation. We conclude that type I IFN can protect cancer cells from T cell-mediated cytotoxicity through regulation of Serpinb9. This result helps explain why radiation of tumors can stimulate antitumor immunity yet also result in resistance. It further suggests potential targets for intervention to improve therapy and to predict responses.

Neoantigen vaccines hold great promise in cancer immunotherapy, but the comparative efficacy of different vaccine platforms, particularly in the context of tumor burden (TB), remains insufficiently studied. In this research, we evaluated the safety and therapeutic efficacy of synthetic long peptide and mRNA-based vaccines, both designed to target identical neoantigens across different Lewis Lung Carcinoma (LLC) tumor burdens. We employed the LLC syngeneic mouse model, a widely used preclinical model for aggressive and immunosuppressive tumors. Our findings demonstrated that the mRNA-based vaccine significantly outperformed the peptide-based vaccine in preventing tumor growth in mice with low TB. These results underscore the potential of mRNA vaccines as a more effective approach for treating aggressive tumors, contributing valuable insights for the future development of neoantigen-based cancer vaccines.