Tumor-draining Lymph Nodes Research Articles

Phenotypical differences of neutrophils patrolling tumour-draining lymph nodes in head and neck cancer

Abstract Background The complexity and heterogeneity of neutrophils are recognized, especially their roles in modulating inflammation and cancer immune responses. The detailed functions of neutrophils in human tumour-draining lymph nodes (TDLNs), specifically in the context of head and neck cancer, remain inadequately characterized. Aim This study aims to delineate the phenotypic diversity of neutrophils in TDLNs, non-tumour-draining lymph nodes (nTDLNs) from patients with oral squamous cell carcinoma (OSCC), and to evaluate their correlation with clinical outcomes. Methods A flow cytometry-based investigation. Results Neutrophils manifest a tissue-specific heterogeneity with significant phenotypic differences between compartments. A substantial fraction of neutrophils displayed an activated CD16highCD62Ldim profile in TDLNs, more prominent in patients with advanced T stages, implicating their involvement in the disease’s progression. Notably, the presence of this activated neutrophil phenotype in TDLNs was strongly associated with poorer patient prognosis. Conclusions The study confirms the heterogeneity of neutrophils in human TDLNs, aligning with findings from animal models but extending them to show clinical relevance in human disease. The correlation of neutrophil phenotypes with cancer progression and prognosis emphasizes the importance of these cells in the tumour-microenvironment. The data suggests a future possibility to develop targeted therapies that modulate the neutrophilic response in OSCC.

TMIC-07. TRIPLE COCKTAIL IMMUNOMODULATION OF TUMOR DRAINING LYMPH NODES RESULTS IN SYNERGISTIC REVERSAL OF IMMUNOSUPPRESSION AND IMPROVED OVERALL SURVIVAL IN A MURINE GLIOBLASTOMA MODEL

Abstract Clinical trials involving anti-PD-1 fail to improve survival compared to standard of care in glioblastoma (GBM). Despite the advent of newer immunotherapies such as STING agonists and anti-CSF-1R, combination of these strategies is limited by necessitation of intracranial delivery (STING) or off-target side effects with systemic exposure (anti-CSF-1R + anti-PD-1). We have previously shown that delivery of anti-PD-1 to tumor draining lymph nodes via hydrogels decreases systemic exposure of anti-PD-1 and results in improved therapeutic response compared to systemic delivery. This study assesses the efficacy of combining STING agonist (STING), anti-CSF-1R (aCSF1r), and anti-PD-1 (aPD1) into a hydrogel nanoparticle that targets different components of immunosuppression in tumor draining lymph nodes. CT-2A murine glioma cells were orthotopically implanted into C57BL/6J mice with treatment on day 7 via deep cervical injection of hydrogel polymers loaded with either PBS (control), STING, aCSF1r, aPD1, or a combination of all three immunomodulators. Flow cytometry was used for immunophenotyping and Kaplan-Meier survival curves were generated to assess therapeutic efficacy. Each treatment strategy targeted a different component of immunosuppression. Compared to controls, aCSF1r resulted in the greatest decrease of immunosuppressive myeloid cells in the brain (P=0.0017) and cervical lymph nodes (P=0.0008); STING resulted in the greatest activation of intracranial dendritic cells (P=0.0001); and aPD1 resulted in the highest level of T cell activation of the monotherapies (P=0.0008) but also caused compensatory upregulation of regulatory T cells in the brain (P=0.0176). Combination therapy resulted in the greatest decrease in immunosuppression with increased T cell IFN-y production and dendritic cell activation, with resultant improvement in median and overall survival (P=0.0006). Triple combination therapy to tumor draining lymph nodes that specifically targets multiple components of immunosuppression is a safe and efficacious treatment that can be translated to clinical application.

CTIM-13. D2C7-IT IN COMBINATION WITH AN FC-ENGINEERED ANTI-HUMANCD40 MONOCLONAL ANTIBODY (2141-V11) ADMINISTERED INTRATUMORALLY VIA CONVECTION-ENHANCED DELIVERY (CED) FOLLOWED BY CERVICAL PERILYMPHATIC INJECTIONS (CPLIS) OF 2141-V11 IN PATIENTS WITH RECURRENT GLIOBLASTOMA (RGBM)

Abstract BACKGROUND The combination of D2C7-IT, an antibody toxin conjugate, and anti-mouseCD40 via CED generates robust antitumor immunity by killing tumor cells, activating microglia, reversing glioma-associated microglia and macrophage (GAMM)-mediated immune suppression, and empowering polyfunctional tumor antigen-specific CD8 T cells in glioma models. In addition to reprogramming GAMMs, anti-CD40 also activated dendritic cells and induced glioma-specific T cell responses in tumor draining lymph nodes. After establishing the phase 2 dose of the combination of D2C7-IT+2141-V11 when infused via CED, we initiated an assessment of the safety and efficacy of the combination of D2C7-IT+2141-V11 CED followed by repeated subcutaneous CPLIs of 2141-V11 ipsilateral to the tumor in patients with rGBM, IDH wild-type (NCT04547777). METHODS Eligible are adult patients with solitary supratentorial rGBM; ≥4weeks after chemotherapy, bevacizumab, or investigational agent; adequate organ function; and KPS ≥70%. Patients are treated with D2C7-IT (166μg) and 2141-V11 (3mg), both infused via CED, followed by CPLI of 2141-V11 at 2mg at weeks 2 and 4 post D2C7-IT+2141-V11 CED, followed by every 3 weeks for 1 year and then every 4-6 weeks. RESULTS As of May 22, 2024, 18 patients with rGBM (median age of 54) were treated with D2C7-IT+2141-V11 CED followed by CPLIs of 2141-V11. 83% patients remain alive (range 3.3-14.8 months from start of D2C7-IT CED infusion) with a median of 8 CPLIs of 2141-V11 received (range 3-19). No study related grade 4 or 5 adverse events (AEs) were observed. Grade 3 AEs related to D2C7-IT and/or 2141-V11 include one each of hydrocephalus, headache, seizure, and cerebral edema. AEs related exclusively to CPLIs of 2141-V11 consist of grade 1 injection site reaction (n=16). CONCLUSIONS The addition of CPLIs of 2141-V11 post CED of D2C7-IT+2141-V11 is feasible and has shown an acceptable toxicity profile. The safety and efficacy results will be updated.

TMIC-63. DUAL IMMUNE CHECKPOINT BLOCKADE IN A NOVEL EGFR-DRIVEN MURINE GLIOMA MODEL INCREASES PERIPHERAL T CELL PROLIFERATION BUT FAILS TO PROLONG SURVIVAL

Abstract Patients with glioblastoma (GBM) survive only 15-18 months following standard-of-care surgical resection and radio-chemotherapy. Immune checkpoint blockade (ICB), effective in extracranial cancers, failed to improve survival in patients with recurrent GBM in a multi-center phase III clinical trial. As such, we are interested in the molecular mechanisms that drive ICB resistance. Oncogenic EGFR activation is observed in ~50% of GBMs and is associated with immunosuppression; however, whether EGFR signaling impairs T cell function in GBM resulting in limited ICB efficacy remains unclear. Therefore, we leveraged the novel MADR-mEGFRvIII glioma model, which expresses murine EGFRvIII under a tetracycline-off system, to investigate the relationship between EGFR activation and responsiveness to dual anti-PD-1 and anti-CTLA-4 (10mg/kg) in vivo. Mice were bled prior to enrollment and weekly thereafter, which confirmed anti-PD-1-PD-1 binding via competitive flow cytometric assay. Following two weeks on treatment, animals were euthanized for immunophenotypic analyses of tumors and tumor-draining lymph nodes. These analyses revealed a significantly increased prevalence of peripheral CD11b+ myeloid cells and enhanced peripheral T cell proliferation, indicated by Ki67 positivity, after treatment with dual ICB compared to isotype control. However, these shifts did not translate to effective ICB-mediated recruitment of peripheral T cells to the tumor microenvironment and no significant survival benefit was observed (p=0.68, n=15/treatment arm). Given our previous findings that EGFR ablation via doxycycline enhanced intratumoral T cell infiltration, we hypothesize the rational combination of EGFR-targeted therapy and ICB will have a synergistic effect; thus, future directions include a study combining the two treatment regimens consisting of a lead-in with doxycycline to increase intratumoral T cell trafficking, followed by treatment with dual ICB. This work will provide insight into the interplay between oncogenic signaling and immunosuppression, contributing to the development of novel combination therapies in treating this lethal disease.

Modeling Lymphoma Angiogenesis, Lymphangiogenesis, and Vessel Co-Option, and the Effects of Inhibition of Lymphoma–Vessel Interactions with an αCD20-EndoP125A Antibody Fusion Protein

Lymphoma growth, progression, and dissemination require tumor cell interaction with supporting vessels and are facilitated through tumor-promoted angiogenesis, lymphangiogenesis, and/or lymphoma vessel co-option. Vessel co-option has been shown to be responsible for tumor initiation, metastasis, and resistance to anti-angiogenic treatment but is largely uncharacterized in the setting of lymphoma. We developed an in vitro model to study lymphoma–vessel interactions and found that mantle cell lymphoma (MCL) cells co-cultured on Matrigel with human umbilical vein (HUVEC) or human lymphatic (HLEC) endothelial cells migrate to and anneal with newly formed capillary-like (CLS) or lymphatic-like (LLS) structures, consistent with lymphoma–vessel co-option. To inhibit this interaction, we constructed an antibody fusion protein, αCD20-EndoP125A, linking mutant anti-angiogenic endostatin (EndoP125A) to an αCD20-IgG1-targeting antibody. αCD20-EndoP125A inhibited both CLS and LLS formation, as well as MCL migration and vessel co-option. Lymphoma vessel co-option requires cell migration, which is regulated by chemokine–chemokine receptor interactions. CXCL12 and its receptor, CXCR4, are highly expressed by both endothelial cells forming CLS and by MCL cells during vessel co-option. αCD20-EndoP125A suppressed expression of both CXCL12 and CXCR4, which were required to facilitate CLS assembly and vessel co-option. We also tested αCD20-EndoP125A effects in vivo using an aggressive murine B cell lymphoma model, 38c13-hCD20, which demonstrated rapid growth and dissemination to tumor-draining lymph nodes (TDLNs) and the spleen, lung, and brain. The pattern of lymphoma distribution and growth within the lung was consistent with vessel co-option. As predicted by our in vitro model, αCD20-EndoP125A treatment inhibited primary tumor growth, angiogenesis, and lymphangiogenesis, and markedly reduced the number of circulating tumor cells and lymphoma dissemination to TDLNs and the lungs, spleen, and brain. αCD20-EndoP125A inhibited lymphoma vessel co-option within the lung. Marked inhibition of MCL primary tumor growth and dissemination were also seen using an MCL xenograft model. The ability of αCD20-EndoP125A to inhibit angiogenesis, lymphangiogenesis, and lymphoma vessel co-option provides a novel therapeutic approach for inhibition of lymphoma progression and dissemination.

Therapeutic Immunomodulation of Tumor-Lymphatic Crosstalk via Intratumoral Immunotherapy.

Intra- and peritumoral lymphatics and tumor-draining lymph nodes play major roles in mediating the adaptive immune response to cancer immunotherapy. Despite this, current paradigms of clinical cancer management seldom seek to therapeutically modulate tumor-lymphatic immune crosstalk. This review explores recent developments that set the stage for how this regulatory axis can be therapeutically manipulated, with a particular emphasis on tumor-localized immunomodulation. Building on this idea, the nature of tumor-lymphatic immune crosstalk and relevant immunotherapeutic targets and pathways are reviewed, with a focus on their translational potential. Engineered drug delivery systems that enhance intratumoral immunotherapy by improving drug delivery to both the tumor and lymph nodes are also highlighted.

A biomimetic solution, albumin-doxorubicin molecular complex, targeting tumor and tumor-draining lymph nodes.

Chemotherapy-induced immunologic cell death is haunted by the non-specific distribution of chemotherapeutic drugs and insignificant immune activation effects, which render efforts to inhibit the distant metastasis of tumors frustrated. Given the pivotal role that lymph nodes play in tumor metastasis, it is of vital importance whether the drug delivery to tumor-draining lymph nodes (TDLNs) succeeds. In the current study, we developed a doxorubicin-albumin complex (DOX-HSA) solution with the specific ability to simultaneously target the primary tumor and the TDLNs. DOX-HSA could effectively activate and amplify the immunogenic cell death (ICD) effect in both the tumor tissues and the TDLNs, resulting in increased release of damage-associated molecular patterns (DAMPs), which further promoted phagocytosis and maturation of dendritic cells (DCs), stimulated activation of CD8+T cells, and then significantly enhanced the therapeutic effects of doxorubicin on orthotopic 4T1 tumor-bearing model mice. Therefore, the DOX-HSA solution demonstrated a more prominent ability to control cancer cells and curb metastasis, as well as improved security by reducing cardiotoxicity and myelosuppression toxicity of doxorubicin itself. This DOX-HSA strengthened the synergistic anti-tumor effects based on the ICD effect in combination with traditional chemotherapy, thus providing promising prospects for clinical application.

Differentiation fate of a stem-like CD4 T cell controls immunity to cancer.

The T cell response to cancer controls disease progression and response to immunotherapy1-3. Despite extensive knowledge regarding CD8 T cells, how CD4 T cells contribute to this process is less well understood. Here we identified a population of PD1+TCF1+ CD4 T cells with stem-like properties that are capable of self-renewal and differentiation into canonical CD4 effector cells. Primarily residing in tumour-draining lymph nodes (TDLNs), these tumour-specific CD4 T cells are restricted by T regulatory (Treg) cells to a stem-like fate that predominantly generated induced Treg (iTreg) cells, limiting effector CD8 T cell responses to the tumour. By contrast, upon Treg depletion, stem-like CD4 T cells differentiated into T helper 1 (TH1) cells, and via IFNγ production induced robust effector differentiation from TCF1+ CD8 T cells in TDLNs, a state we defined as 'active'. Notably, enforcing TBET expression in transferred stem-like CD4 T cells was sufficient to overcome the established restricted T cell state. Despite the presence of Treg cells, endogenous stem-like CD4 T cells actively generated TH1 cells, which were required to restore TDLN effector CD8 T cell differentiation, enhance tumour control and rescue response to immunotherapy. In agreement, TH1 differentiation in patients with kidney cancer predicted successful immunotherapy responses and improved progression-free survival. Together, these findings identify a stem-like CD4 T cell population that through alternative differentiation fates controls the switch between restricted and active T cell states with implications for cancer immunotherapies.

Enhanced Tumor Immunotherapy by Triple Amplification Effects of Nanomedicine on the STING Signaling Pathway in Dendritic Cells.

Insufficient activation of stimulator of interferon genes (STING) signaling pathway in tumor-associated dendritic cells limits the efficiency of tumor immunotherapy. Herein, the "three-in-one" IAHA-LaP/siPTPN6 NPs containing lanthanum ions (La3+), cGAMP, and PTPN6 siRNA are developed for triple amplification of the STING pathway. In vitro results demonstrate that La3+ significantly promotes cGAMP-mediated activation of the STING pathway by enhancing the phosphorylation of STING, TBK1, IRF3, and NF-κB p65. Moreover, the IAHA-LaP/siPTPN6 NPs further significantly enhance the phosphorylation of STING and NF-κB p65 and augment K63-linked ubiquitination of STING protein via siPTPN6-mediated downregulation of SHP-1 protein. Furthermore, NPs improve the secretion of IFNβ (2.4-fold), IL-6 (1.5-fold), and TNF-α (1.4-fold), thereby promoting DCs maturation compared to the mixture of La3+ and cGAMP. In vivo results show that the IAHA-LaP/siPTPN6 NPs remarkably inhibit primary tumor growth by increasing the percentage of mature DCs in tumor-draining lymph nodes, polarizing M2/M1 phenotype in TME, and promoting the infiltration of CD8+T cells into tumors. Moreover, these NPs dramatically prevent the growth of distal tumor by inducing systemic anti-tumor immunity and generating a long-term anti-tumor memory for protection against tumor recurrence in mice bearing bilateral B16F10. These IAHA-LaP/siPTPN6 NPs may offer a promising platform for robust anti-tumor immune responses.

P4.07E.08 Single-Cell TCR Barcoding and RNA Sequencing Illuminate Distinct Tumor-Draining Lymph Node T Cell Responses to Neoadjuvant PD-1 Blockade

P4.07E.08 Single-Cell TCR Barcoding and RNA Sequencing Illuminate Distinct Tumor-Draining Lymph Node T Cell Responses to Neoadjuvant PD-1 Blockade

H2O2 Self-Supplying CaO2 Nanoplatform Induces Ca2+ Overload Combined with Chemodynamic Therapy to Enhance Cancer Immunotherapy.

Integrating chemodynamic therapy (CDT) with Ca2+ overload offers a potent strategy for enhancing cancer immunotherapy. However, the effectiveness of this approach is significantly constrained by the scarce availability of H2O2 in solid tumors. Here, we engineered a nanoplatform based on CaO2 nanoparticles (NPs) capable of encapsulating curcumin (CUR) and self-supplying H2O2 for synergistic CDT-augmented antitumor immunotherapy (CaO2@CUR@ZIF-Cu, denoted as CCZC). In the acidic tumor microenvironment, CCZC disintegrated to release CUR and copper(II) ions (Cu2+), revealing the core CaO2 NPs. CDT was amplified by escalating hydroxyl radical (•OH) production through a Fenton-like reaction mediated by H2O2 from the hydrolysis of CaO2 NPs. Ca2+ sourced from CaO2 NPs and CUR, an initiator of Ca2+ overload, induced Ca2+ overload in tumor cells, thereby promoting apoptosis. Subsequently, apoptotic tumor cells released tumor-associated antigens and pro-inflammatory cytokines, triggering adaptive immune responses and enhancing antitumor immunotherapy effects. In vivo experiments demonstrated that the intratumoral administration of CCZC displayed significant inhibitory effects, with an inhibition rate of up to 78% on B16-OVA-tumor-bearing mice compared to untreated. Moreover, an elevated proportion of mature dendritic cells were observed in the tumor-draining lymph nodes, along with an increase in cytotoxic T lymphocytes in the spleen. These findings suggest that our engineered nanoplatform effectively curtailed tumor growth via enhanced cancer immunotherapy by synergizing Ca2+ overload and CDT, proposing a novel strategy for synergistic cancer treatment.

Prognostic value of T regulatory cells and immune checkpoints expression in tumor-draining lymph nodes for oral squamous cell carcinoma.

Forty-nine OSCC patients were enrolled. One TDLN per patient was analysed using flow cytometry to profile immune-checkpoint expression (PD-1, CTLA-4, TIGIT, TIM-3, LAG-3) and other markers such as CD69, CXCR5 on CD4+, CD8+, and Tregs. Disease-free survival (DFS) and overall survival (OS) were assessed. According to multivariate analysis, elevated levels of FoxP3+CD4+ and TIGIT+CD8+ cells in TDLNs correlated with significantly worse DFS, while high CXCR5+CD4+ levels were associated with better DFS. Notably, the expression of immune checkpoints on T cells within TDLNs showed significant associations with recurrence status. Patients experiencing recurrence exhibited heightened levels of T regulatory cells, CD4+PD-1+ and CD4+CTLA-4+, cells in TDLNs. Survival multivariate analyses revealed that T status emerged as an independent predictor of OS. The findings highlight the critical role of TDLNs in the immune microenvironment of OSCC and establish immune checkpoint expression on T cells as promising prognostic biomarkers. These insights upgrade the prognostic framework for OSCC and pave the way for individualized therapeutic strategies. The prognostic significance of TDLNs and a high expression of immune checkpoint inhibitors is a compelling argument for the adoption of neoadjuvant immunotherapy.

Node-sparing modified short-course Radiotherapy Combined with CAPOX and Tislelizumab for locally Advanced MSS of Middle and low rectal Cancer (mRCAT): an open-label, single-arm, prospective, multicentre clinical trial

BackgroundNeoadjuvant chemoradiotherapy followed by total mesorectal excision is a standard treatment for locally advanced rectal cancer. Mismatch repair-deficient locally advanced rectal cancer (LARC) was highly sensitive to PD-1 blockade. However, most rectal cancers are microsatellite stable (MSS) or mismatch repair-proficient (pMMR) subtypes for which PD-1 blockade is ineffective. Radiation can trigger the activation of CD8 + T cells, further enhancing the responses of MSS/pMMR rectal cancer to PD-1 blockade. Radioimmunotherapy offers a promising therapeutic modality for rectal cancer. Progenitor T exhausted cells are abundant in tumour-draining lymph nodes and play an important role in immunotherapy. Conventional irradiation fields include the mesorectum and regional lymph nodes, which might cause considerable damage to T lymphocytes and radiation-induced fibrosis, ultimately leading to a poor response to immunotherapy and rectal fibrosis. This study investigated whether node-sparing modified short-course irradiation combined with chemotherapy and PD-1 blockade could be effective in patients with MSS/ pMMR LARC.MethodsThis was a open-label, single-arm, multicentre, prospective phase II trial. 32 LARC patients with MSS/pMMR will receive node-sparing modified short-course radiotherapy (the irradiated planned target volume only included the primary tumour bed but not the tumour-draining lymph nodes, 25 Gy/5f, 5 Gy/f) followed by CAPOX and tislelizumab. CAPOX and tislelizumab will be started two days after the completion of radiotherapy: oxaliplatin 130 mg/m2 intravenous infusion, day 1; capecitabine 1000 mg/m2 oral administration, days 1–14; and tislelizumab 200 mg, intravenous infusion, day 1. There will be four 21-day cycles. TME will be performed at weeks 14–15. We will collect blood, tumour, and lymphoid specimens; perform flow cytometry and in situ multiplexed immunofluorescence detection; and analyse the changes in various lymphocyte subsets. The primary endpoint is the rate of pathological complete response. The organ preservation rate, tumour regression grade, local recurrence rate, disease-free survival, overall survival, adverse effects, and quality of life will also be analysed.DiscussionIn our research, node-sparing modified radiotherapy combined with immunotherapy probably increased the responsiveness of immunotherapy for MSS/pMMR rectal cancer patients, reduced the occurrence of postoperative rectal fibrosis, and improved survival and quality of life. This is the first clinical trial to utilize a node-sparing radiation strategy combined with chemotherapy and PD-1 blockade in the neoadjuvant treatment of rectal cancer, which may result in a breakthrough in the treatment of MSS/pMMR rectal cancer.Trial registrationThis study was registered at www.clinicaltrials.gov. Trial registration number: NCT05972655. Date of registration: 31 July 2023.

The novel and potent CD40 agonist KHK2840 augments the antitumor efficacy of anti-PD-1 antibody and paclitaxel.

Lack of tumor-reactive cytotoxic T lymphocytes (CTLs)limits the antitumor efficacy of immune checkpoint inhibitors (ICIs). CD40 agonists have been expected to overcome this limitation by generating tumor-reactive CTLs. However, the clinical efficacy of CD40 agonistic antibodies is not as good as in non-clinical studies. The novel human CD40 (hCD40) agonist KHK2840 is a fully human anti-CD40 IgG2 agonistic antibody that is Fc-engineered to minimize complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity. Compared to other hCD40 agonists, KHK2840 exhibited the most potent hCD40 agonistic signal in tumor-bearing hCD40 transgenic mice and human peripheral blood B cells. Moreover, KHK2840 enhanced the antitumor efficacy of the antiprogrammed cell death 1 antibody and paclitaxel. Comprehensive immune profiling revealed that the antitumor immune response of the triple combination involved tumor-draining lymph nodes in addition to tumor microenvironments. This suggests that a coordinated antitumor immune response between tumors and lymph nodes may underlie the synergistic antitumor efficacy of the triple combination therapy. Finally, a toxicology study in cynomolgus monkeys demonstrated that KHK2840 activated the CD40 signal with tolerable toxicological properties. These results indicate that KHK2840 is a novel and potent hCD40 agonistic antibody for cancer immunotherapy, which is expected to augment the antitumor efficacy of ICIs and chemotherapy.

A Novel Chimeric Oncolytic Virus Mediates a Multifaceted Cellular Immune Response in a Syngeneic B16 Melanoma Model

Background/Objectives: Oncolytic virotherapy is a promising approach in cancer immunotherapy. We have previously described a recombinant hybrid oncolytic virus (OV), VSV-NDV, which has a favorable safety profile and therapeutic immunogenicity, leading to direct oncolysis, abscopal effects, and prolonged survival in syngeneic in vivo tumor models. While OVs are known to mediate systemic anti-tumor immune responses, the detailed characterization of local and systemic immune responses to fusogenic oncolytic virotherapy remains unexplored. Methods and Results: We analyzed immune cell compartments in the spleen, blood, tumor-draining lymph nodes (TDLNs), and tumors over the course of VSV-NDV therapy in a bilateral syngeneic melanoma mouse model. Our results revealed significant local infiltration and activation of T lymphocytes in tumors and globally in the blood and spleen. Notably, in vivo CD8+ T cell depletion led to complete abrogation of the tumor response, highlighting the crucial role of T cells in promoting the therapeutic effects of oncolytic VSV-NDV. In vitro co-culture experiments enabled the interrogation of human immune cell responses to VSV-NDV-mediated oncolysis. Human peripheral blood mononuclear cells (PBMCs) were efficiently stimulated by exposure to VSV-NDV-infected cancer cells, which recapitulates the in vivo murine findings. Conclusions: Taken together, these data characterize a broad anti-tumor immune cell response to oncolytic VSV-NDV therapy and suggest that CD8+ T cells play a decisive role in therapeutic outcome, which supports the further development of this chimeric vector as a multimechanistic immunotherapy for solid cancers.

Tumor draining lymph nodes connected to cold triple-negative breast cancers are characterized by Th2-associated microenvironment

Tumor draining lymph nodes (TDLN) represent a key component of the tumor-immunity cycle. There are few studies describing how TDLNs impact lymphocyte infiltration into tumors. Here we directly compare tumor-free TDLNs draining “cold” and “hot” human triple negative breast cancers (TDLNCold and TDLNHot). Using machine-learning-based self-correlation analysis of immune gene expression, we find unbalanced intranodal regulations within TDLNCold. Two gene pairs (TBX21/GATA3-CXCR1) with opposite correlations suggest preferential priming of T helper 2 (Th2) cells by mature dendritic cells (DC) within TDLNCold. This is validated by multiplex immunofluorescent staining, identifying more mature-DC-Th2 spatial clusters within TDLNCold versus TDLNHot. Associated with this Th2 priming preference, more IL4 producing mast cells (MC) are found within sinus regions of TDLNCold. Downstream, Th2-associated fibrotic TME is found in paired cold tumors with increased Th2/T-helper-1-cell (Th1) ratio, upregulated fibrosis growth factors, and stromal enrichment of cancer associated fibroblasts. These findings are further confirmed in a validation cohort and public genomic data. Our results reveal a potential role of IL4+ MCs within TDLNs, associated with Th2 polarization and reduced immune infiltration into tumors.

Proximity-dependent labeling identifies dendritic cells that drive the tumor-specific CD4+ T cell response.

Dendritic cells (DCs) are uniquely capable of transporting tumor antigens to tumor-draining lymph nodes (tdLNs) and interact with effector T cells in the tumor microenvironment (TME) itself, mediating both natural antitumor immunity and the response to checkpoint blockade immunotherapy. Using LIPSTIC (Labeling Immune Partnerships by SorTagging Intercellular Contacts)-based single-cell transcriptomics, we identified individual DCs capable of presenting antigen to CD4+ T cells in both the tdLN and TME. Our findings revealed that DCs with similar hyperactivated transcriptional phenotypes interact with helper T cells both in tumors and in the tdLN and that checkpoint blockade drugs enhance these interactions. These findings show that a relatively small fraction of DCs is responsible for most of the antigen presentation in the tdLN and TME to both CD4+ and CD8+ tumor-specific T cells and that classical checkpoint blockade enhances CD40-driven DC activation at both sites.

A size-switchable nanocluster remodels the immunosuppressive microenvironment of tumor and tumor-draining lymph nodes for improved cancer immunotherapy

Remodeling the immunosuppressive tumor microenvironment (TME) by immunomodulators has been well studied in the past years. However, strategies that enable concurrent modulation of both the immunosuppressive TME and tumor-draining lymph nodes (TDLNs) are still in the infancy. Here, we report a pH-sensitive size-switchable nanocluster, SPN-R848, to achieve simultaneous accumulation in tumor and TDLNs for immune activation. SPN-R848 with original size around 150 nm was formed by self-assembly of resiquimod (R848)-conjugated polyamidoamine (PAMAM) derivative, which could disintegrate into its small constituents (～ 8 nm) upon exposure to tumor acidity. The size reduction not only enhanced their accumulation and perfusion in the primary tumor, but promoted their transport and distribution in TDLNs. Accordingly, SPN-R848 remarkably remodeled the immunosuppressive TME by polarizing M2 to M1 macrophages and activated dendritic cells (DCs) in TDLNs, which synergistically facilitated the production and stimulation of cytotoxic T cells, and inhibited tumor growth in breast cancer and melanoma mouse models. Our study suggests that co-activation of immune microenvironments in both tumor and TDLNs may represent a promising direction to elicit strong antitumor immunity.

Inhibitory Fcγ receptor deletion enhances CD8 T cell stemness increasing anti-PD-1 therapy responsiveness against glioblastoma

BackgroundCertain cancers present challenges for treatment because they are resistant to immune checkpoint blockade (ICB), attributed to low tumor mutational burden and the absence of T cell-inflamed features. Among these,...

PD-L1 in Melanoma and Extracellular Vesicles Promotes Local and Regional Immune Suppression through M2-like Macrophage Polarization

Tumor-associated macrophages (TAMs) exhibit dual roles in tumor progression. TAMs are known to induce PD-L1 expression in cancer cells. However, the regulatory effects of PD-L1 in melanoma cells on TAM phenotypical switching remain underexplored. Herein, our findings indicated that CD163 and MRC1 levels were significantly elevated in metastatic melanomas compared to primary melanomas, correlating with CD274 expression and predicted patient clinical outcomes. To study the mechanisms regulating M2-like polarization, PD-L1 was knocked out in both YUMM1.7 and B16-F10 melanoma cells. The data revealed that knocking out PD-L1 (PD-L1KO) in melanoma resulted in a decelerated in vivo growth rate, accompanied by a significantly increased M1/M2 ratio, more dendritic cells, and enhanced activation of CD8+ T cells compared to wild-type (WT) melanoma cells. These alterations were associated with decreased expression of M2-associated chemokines (CCL2, CCL3, and CXCL2) and cytokines (IL6, IL10, and TGFβ1). Mice harboring PD-L1KO melanomas exhibited elevated levels of CD8+ T cells in both the tumor-draining lymph nodes and the bloodstream, compared to mice with PD-L1WT melanomas. Treatment with extracellular vesicles (EVs) derived from PD-L1KO melanoma resulted in a reduced tumor growth rate and fewer M2-like macrophages in the tumors compared to EVs from PD-L1WT melanomas. Therefore, our data suggest that PD-L1 in melanoma and melanoma-derived EVs induces M2-like polarization, contributing to local and regional immune suppression.