T-cell Priming Research Articles

Antigen epitope-TLR7/8a conjugate as self-assembled carrier-free nanovaccine for personalized immunotherapy

Epitope-based vaccine is a promising personalized cancer immunotherapy; however, a simple and effective approach for its bulk manufacturing is challenging. Current vaccination strategies complicate the process by introducing unnecessary components such as additional delivery carriers, and assembly units. Herein, a type of toll-like receptor 7/8 agonist-epitope conjugate (termed as TLR7/8a-epitope) has been developed as a self-assembled and carrier-free nano vaccine platform, which effectively introduces the antigen and adjuvant with maximum precision, resulting in significantly enhanced dendritic cells (DCs) activation through the MyD88-dependent TLR signaling pathway. TLR7/8a-epitope nanovaccine can prolong the local retention and increase drainage efficiency into the lymph node, eliciting a significantly higher level of CD8 T-cell immunity than those of conventional vaccine formulations. The immunization with TLR7/8a-epitope nanovaccine in mice can not only resist the invasion of B16 cancer cells, but also produce significant therapeutic effects against established B16 melanoma tumors. Therefore, the TLR7/8a-epitope nanovaccine, developed by the direct chemical conjugation of antigen peptide with immunoadjuvant, has great advantages of clear and leanest compositions, controllable and definite preparation process, and remarkable therapeutic effects, representing a new appraoch for personalized cancer immunotherapy. Statement of significanceHerein, a kind of toll-like receptor 7/8 agonist-epitope conjugate was developed and spontaneously self-assemble into nanostructure in aqueous solution without the use of any additional constituents, which can be termed as unique carrier-free nanovaccine platform, providing effectually the leanest vaccine components with maximally and precisely loading of antigen and adjuvant.Significantly, the nanovaccine augmented the immunogenicity of antigenic peptide by increasing DCs activation through MyD88-mediated TLR signaling pathways and promoting T-cell priming. Moreover, nanovaccines could prolong the local retention and further increase the efficiency of drainage into dLNs, which was contributing to efficient initiation of epitope-specific memory and effector T-cell immune responses, leading to effective prophylactic and therapeutic antitumor effects.

Disruption of c-MYC Binding and Chromosomal Looping Involving Genetic Variants Associated With Ankylosing Spondylitis Upstream of the RUNX3 Promoter.

Background: Ankylosing Spondylitis (AS) is a common form of inflammatory spinal arthritis with a complex aetiology and high heritability, involving more than 100 genetic associations. These include several AS-associated single nucleotide polymorphisms (SNPs) upstream of RUNX3, which encodes the multifunctional RUNT-related transcription factor (TF) 3. The lead associated SNP rs6600247 (p = 2.6 × 10−15) lies ∼13kb upstream of the RUNX3 promoter adjacent to a c-MYC TF binding-site. The effect of rs6600247 genotype on DNA binding and chromosome looping were investigated by electrophoretic mobility gel shift assays (EMSA), Western blotting-EMSA (WEMSA) and Chromosome Conformation Capture (3C). Results: Interrogation of ENCODE published data showed open chromatin in the region overlapping rs6600247 in primary human CD14+ monocytes, in contrast to the Jurkat T cell line or primary human T-cells. The rs6600247 AS-risk allele is predicted to specifically disrupt a c-MYC binding-site. Using a 50bp DNA probe spanning rs6600247 we consistently observed reduced binding to the AS-risk “C” allele of both purified c-MYC protein and nuclear extracts (NE) from monocyte-like U937 cells. WEMSA on U937 NE and purified c-MYC protein confirmed these differences (n = 3; p < 0.05). 3C experiments demonstrated negligible interaction between the region encompassing rs6600247 and the RUNX3 promoter. A stronger interaction frequency was demonstrated between the RUNX3 promoter and the previously characterised AS-associated SNP rs4648889. Conclusion: The lead SNP rs6600247, located in an enhancer-like region upstream of the RUNX3 promoter, modulates c-MYC binding. However, the region encompassing rs6600247 has rather limited physical interaction with the promoter of RUNX3. In contrast a clear chromatin looping event between the region encompassing rs4648889 and the RUNX3 promoter was observed. These data provide further evidence for complexity in the regulatory elements upstream of the RUNX3 promoter and the involvement of RUNX3 transcriptional regulation in AS.

Managing Resistance to Immune Checkpoint Inhibitors in Lung Cancer: Treatment and Novel Strategies.

A proportion of patients with lung cancer experience long-term clinical benefit with immune checkpoint inhibitors (ICIs). However, most patients develop disease progression during treatment or after treatment discontinuation. Definitions of immune resistance are heterogeneous according to different clinical and biologic features. Primary resistance and acquired resistance, related to tumor-intrinsic and tumor-extrinsic mechanisms, are identified according to previous response patterns and timing of occurrence. The clinical resistance patterns determine differential clinical approaches. To date, several combination therapies are under development to delay or prevent the occurrence of resistance to ICIs, including the blockade of immune coinhibitory signals, the activation of those with costimulatory functions, the modulation of the tumor microenvironment, and the targeting T-cell priming. Tailoring the specific treatments with distinctive biologic resistance mechanisms would be ideal to improve the design and results of clinical trial. In this review, we reviewed the available evidence on immune resistance mechanisms, clinical definitions, and management of resistance to ICIs in lung cancer. We also reviewed data on novel strategies under investigation in this setting.

A Sampling of Highlights from the Literature: Article Recommendations from Our Deputy and Senior Editors.

Nanotubes mediate mitochondrial transfer to tumor cells (by Michael Ströck via Wikimedia Commons)Tumors use various mechanisms to escape immune detection in the tumor microenvironment (TME). Saha et al. show that tumor cells form nanotubes that physically interact with immune cells in the breast cancer TME. Mitochondria are then transferred from immune cells to tumor cells, leading to tumor metabolic enhancement and loss of specific immune-cell subsets needed for antitumor responses. Targeting nanotube formation reversed these effects and when combined with immune checkpoint blockade, resulted in improved outcomes in a murine breast cancer model. The data highlight a new immune “hijacking” mechanism that tumors cells could use to promote cancer progression.Saha T, …, Sengupta S. Nat Nanotechnol 2021 Nov 18. DOI: 10.1038/s41565-021-01000-4.Lung cancers can become infiltrated by dysfunctional CD8+ T cells (by Eric Synder via NCI Visuals Online)Many non–small cell lung cancers (NSCLC) infiltrated with CD8+ T cells are resistant to immune checkpoint blockade. In an orthotopic NSCLC model, Horton et al. find lung tumors are infiltrated with CD8+ T cells but do not respond to combination anti–CTLA-4 and anti–PD-1. The CD8+ T cells have a dysfunctional phenotype molecularly distinct from T-cell exhaustion. The dysfunctional phenotype is induced during T-cell priming in lymph nodes and characterized by failure to acquire an effector program. Treatment with IL2 and IL12 restores effector T-cell differentiation. As CD8+ T cells from NSCLC patients have an analogous gene expression program of dysfunction, the data have translational potential.Horton BL, …, Spranger S. Sci Immunol 2021 Oct 29;6:eabi8800.γδ T cells infiltrate many tumor types and are highly cytotoxic (from Fig. 5C of Gherardin et al., Cancer Immunol Res 2021)Antibodies specific for butyrophilin 3A (BTN3A) can induce Vγ9Vδ2 T–cell cytolytic activity. De Gassart et al. have engineered a humanized BTN3A-specific antibody (ICT01) that induces Vγ9Vδ2 T–cell killing of several human tumor cell lines and primary tumor cells in vitro and xenografted tumors in mice following Vγ9Vδ2 T–cell transfer. Preliminary data from six patients with solid tumors administered ICT01 in the phase I/IIa EVICTION clinical trial (NCT04243499) show the antibody can be well tolerated, has caused no major safety events, and appears to induce broad immune-cell infiltration of tumors. Further studies should reveal the full potential of this strategy.De Gassart A, …, Frohna P. Sci Transl Med 2021 Oct 20;13:eabj0835.Histamine can suppress antitumor responses (by NEUROtiker via Wikimedia Commons)Identification of obstacles to effective antitumor immunity sometimes reveals surprising participants. Li et al. find that histamine, a mediator of allergic responses, plays an inhibitory role in antitumor responses. Histamine is frequently found in the tumor microenvironment and can activate macrophages via histamine receptor H1 (HRH1), promoting polarization to a suppressive M2-like phenotype and upregulation of the inhibitory checkpoint VISTA. Such macrophages suppress T-cell responses and promote tumor growth and therapy resistance. Knockout of HRH1 or use of antihistamines rescues experimental antitumor responses. The study highlights a role for histamine in modulating antitumor responses and suggests antihistamines as potential adjuvants for cancer immunotherapies.Li H, …, Yu D. Cancer Cell 2021 Nov 24. DOI: 10.1016/j.ccell.2021.11.002.PC-CAR T cells could unlock the immunotherapeutic potential of intracellular antigens (by Paris on Ponce &amp; Le Maison Rouge via Flickr)Developing chimeric receptor (CAR) T cells specific for intracellular tumor-specific antigens is challenging because these antigens are only available to the immune system as peptides presented on the cell surface by MHC class I. Yarmarkovich et al. overcome this challenge by generating peptide-centric CARs (PC-CAR), which can recognize peptides derived from intracellular oncoproteins presented by multiple HLA types. PC-CAR T cells targeting a peptide from the neuroblastoma oncoprotein PHOX2B selectively kill neuroblastoma cells in vitro and clear tumors transplanted into mice. This study indicates PC-CARs have the potential to unlock the vast number of intracellular oncoproteins as potential targets for CAR T-cell immunotherapy.Yarmarkovich M, …, Maris JM. Nature 2021 Nov 3;599:477–84.TILs accumulate in ovarian cancer islets (from Fig. 1C of Heath et al., Cancer Immunol Res 2021)The tumor microenvironment (TME) factors that regulate exhaustion of CD8+ tumor-infiltrating lymphocytes (TIL) are not fully understood. Duraiswamy et al. found activated, tumor-specific CD8+ TILs concentrated in ovarian cancer islets, where they are closely associated with myeloid antigen-presenting cells (APC) but restrained by PD-1 signaling. Activated islet-resident APCs can provide CD28 costimulation, which is required for exhausted TILs to effectively respond to anti–PD-1 treatment in vitro. These findings suggest that a TME with insufficient CD28 signaling from myeloid APCs could explain poor responses to anti–PD-1 in tumors with substantial TIL populations and that activating the APCs with reagents such as anti-CD40 may rescue therapeutic responses.Duraiswamy J, …, Coukos G. Cancer Cell 2021 Dec 13;39:1623–42.e20.

Streptococcus pneumoniae Impairs Maturation of Human Dendritic Cells and Consequent Activation of CD4+ T Cells via Pneumolysin

Influenza A Virus (IAV), Staphylococcus aureus (staphylococci), and Streptococcus pneumoniae (pneumococci) are leading viral and bacterial causes of pneumonia. Dendritic cells (DCs) are present in the lower respiratory tract. They are characterized by low expression of co-stimulatory molecules, including CD80 and CD86 and high capacity of antigen uptake. Subsequently, DCs upregulate co-stimulatory signals and cytokine secretion to effectively induce T-cell priming. Here, we investigated these processes in response to bacterial and viral single as well as coinfections using human monocyte-derived (mo)DCs. Irrespective of single or coinfections, moDCs matured in response to IAV and/or staphylococcal infections, secreted a wide range of cytokines, and activated CD4⁺, CD8⁺ as well as double-negative T cells. In contrast, pneumococcal single and coinfections impaired moDC maturation, which was characterized by low expression of CD80 and CD86, downregulated expression of CD40, and a mild cytokine release resulting in abrogated CD4⁺ T-cell activation. These actions were attributed to the cholesterol-dependent cytotoxin pneumolysin (Ply). Infections with a ply-deficient mutant resulted in restored moDC maturation and exclusive CD4⁺ T-cell activation. These findings show that Ply has important immunomodulatory functions, supporting further investigations in specific modalities of Ply-DC interplay.

Approaches of the Innate Immune System to Ameliorate Adaptive Immunotherapy for B-Cell Non-Hodgkin Lymphoma in Their Microenvironment.

Simple SummaryThe innate immune checkpoint blockade anti-CD47 antibodies combined with the anti-CD20 antibody rituximab can invigorate macrophage phagocytosis. Generally applied as vaccine adjuvants, TLR9 agonist CpG-oligodeoxynucleotides (CpG-ODNs) stimulate antigen presenting cells (APCs) and plasmacytoid dendritic cells (pDCs) to secrete interferon (IFN)-α/β, which activates natural killer (NK) cells. This innate immune activation and pDC maturation leads to potentiation of the adaptive immune response. In fact, stimulator of interferon gene agonists also induce APCs to secrete IFN-α/β. CpG-ODNs, combined with rituximab or irradiation, demonstrated clinical efficacy in indolent B-cell non-Hodgkin lymphoma patients. Furthermore, patients with mantle cell lymphoma presenting Ig-derived neoantigens and vaccinated with CpG-ODN priming T cells maintained minimal residual disease. Additionally, IFN-α–conditioned DC vaccination as well as 4-1BB and OX40 agonist antibodies activate NK cells as well as T cells. Collectively, these innate activators are promising agents for enhancing anti-B-cell-lymphoma adaptive immunity, such as chimeric antigen receptor-T-cell therapy.A dominant paradigm being developed in immunotherapy for hematologic malignancies is of adaptive immunotherapy that involves chimeric antigen receptor (CAR) T cells and bispecific T-cell engagers. CAR T-cell therapy has yielded results that surpass those of the existing salvage immunochemotherapy for patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL) after first-line immunochemotherapy, while offering a therapeutic option for patients with follicular lymphoma (FL) and mantle cell lymphoma (MCL). However, the role of the innate immune system has been shown to prolong CAR T-cell persistence. Cluster of differentiation (CD) 47-blocking antibodies, which are a promising therapeutic armamentarium for DLBCL, are novel innate immune checkpoint inhibitors that allow macrophages to phagocytose tumor cells. Intratumoral Toll-like receptor 9 agonist CpG oligodeoxynucleotide plays a pivotal role in FL, and vaccination may be required in MCL. Additionally, local stimulator of interferon gene agonists, which induce a systemic anti-lymphoma CD8+ T-cell response, and the costimulatory molecule 4-1BB/CD137 or OX40/CD134 agonistic antibodies represent attractive agents for dendritic cell activations, which subsequently, facilitates initiation of productive T-cell priming and NK cells. This review describes the exploitation of approaches that trigger innate immune activation for adaptive immune cells to operate maximally in the tumor microenvironment of these lymphomas.

Engagement of the costimulatory molecule ICOS in tissues promotes establishment of CD8+ tissue-resident memory T cells

Elevated gene expression of the costimulatory receptor Icos is a hallmark of CD8+ tissue-resident memory (Trm) Tcells. Here, we examined the contribution of ICOS in Trm cell differentiation. Upon transfer into WT mice, Icos-/- CD8+ Tcells exhibited defective Trm generation but produced recirculating memory populations normally. ICOS deficiency or ICOS-L blockade compromised establishment of CD8+ Trm cells but not their maintenance. ICOS ligation during CD8+ Tcell priming did not determine Trm induction; rather, effector CD8+ Tcells showed reduced Trm differentiation after seeding into Icosl-/- mice. IcosYF/YF CD8+ Tcells were compromised in Trm generation, indicating a critical role for PI3K signaling. Modest transcriptional changes in the few Icos-/- Trm cells suggest that ICOS-PI3K signaling primarily enhances the efficiency of CD8+ Tcell tissue residency. Thus, local ICOS signaling promotes production of Trm cells, providing insight into the contribution of costimulatory signals in the generation of tissue-resident populations.

Discovery of Spiro-azaindoline Inhibitors of Hematopoietic Progenitor Kinase 1 (HPK1).

Hematopoietic progenitor kinase 1 (HPK1) is implicated as a negative regulator of T-cell receptor-induced T-cell activation. Studies using HPK1 kinase-dead knock-in animals have demonstrated the loss of HPK1 kinase activity resulted in an increase in T-cell function and tumor growth inhibition in glioma models. Herein, we describe the discovery of a series of small molecule inhibitors of HPK1. Using a structure-based drug design approach, the kinase selectivity of the molecules was significantly improved by inducing and stabilizing an unusual P-loop folded binding mode. The metabolic liabilities of the initial 7-azaindole high-throughput screening hit were mitigated by addressing a key metabolic soft spot along with physicochemical property-based optimization. The resulting spiro-azaindoline HPK1 inhibitors demonstrated improved in vitro ADME properties and the ability to induce cytokine production in primary human T-cells.

Abstract P045: LUMINOS-103: A basket trial evaluating the safety and efficacy of PVSRIPO and PVSRIPO in combination with anti-PD-1/L1 checkpoint inhibitors in patients with advanced solid tumors

Abstract Background: PVSRIPO, a novel intratumoral viral immunotherapy, infects cells via CD155, which is widely expressed on solid tumors and antigen-presenting cells (APC). Infection is lethal in malignant cells, but a unique, activating, nonlethal infection of local APCs yields type-I/III interferon (IFN)-dominant inflammation with subsequent anti-tumor T-cell priming and activation. In preclinical models, PVSRIPO-dependent inflammation upregulated the PD-1/L1 pathway, and greater anti-tumor response was observed with PVSRIPO + anti-PD-1/L1 (αPD-1/L1). Promising clinical activity with PVSRIPO monotherapy was observed in patients (pts) with recurrent glioblastoma and advanced αPD-1–refractory melanoma (Desjardins 2018 NEJM, Beasley 2021 JITC). Collectively, these results warrant further clinical investigation of PVSRIPO ± αPD-1/L1. Trial design, objectives, and eligibility criteria: LUMINOS-103 (NCT04690699) is a phase (Ph) 1/2, open-label, multi-center, single-arm basket trial evaluating repeat administration of PVSRIPO ± αPD-1/L1 in adults with solid tumors. Trial objectives are to assess the safety and tolerability of PVSRIPO monotherapy in each cohort in Ph 1 and the safety, tolerability, and antitumor efficacy of PVSRIPO + αPD-1/L1 in each cohort in Ph 2. The first two study cohorts include pts with muscle-invasive bladder cancer being treated in the neoadjuvant setting (A) and pts with metastatic bladder cancer being treated in the 1st/2nd line setting (B); these cohorts have been described previously (Inman 2021 Virtual AACR Annual Meeting). Cohort C includes pts with resectable, locally advanced head and neck squamous cell carcinoma (HNSCC) being treated in the neoadjuvant setting; Cohort D includes pts with recurrent/metastatic HNSCC with a PD-L1 Combined Positive Score ≥1 being treated in the 1st line setting. Eligibility: HNSCC pts must have histologically or cytologically proven SCC of the oral cavity, oropharynx, hypopharynx, or larynx. All pts must have prior and boosted PV immunization and tumors amenable to injection and biopsy. Key exclusion criteria: Requirement for oxygen supplementation, systemic or intratumoral therapy ≤6 months prior to the first dose of study drug, CNS metastases requiring immediate treatment, systemic immunosuppressive medications ≤4 weeks prior to the first dose of study drug, and severe active comorbidities. Pts who are HIV+, HBV+ or HCV+ are eligible provided they meet certain criteria. Endpoints: Primary endpoints include safety (all), tolerability (all), surgical complication rate (A, C), pathologic treatment effect/response (A, C), and objective response rate (B, D). Secondary endpoints include overall survival (all), pathologic downstaging and relapse-free survival (A, C), duration of response and progression-free survival (B, D), and assessment of tumor/blood biomarkers (all). Citation Format: Brant A. Inman, Matthew I. Milowsky, Raj S. Pruthi, Marshall Posner, Melissa J. Polasek, Shannon R. Morris, Lori Mixson, Kristin Orr, Elizabeth M. H. Woodson, Andrea T. Kelly, W. Garrett Nichols, Arjun V. Balar. LUMINOS-103: A basket trial evaluating the safety and efficacy of PVSRIPO and PVSRIPO in combination with anti-PD-1/L1 checkpoint inhibitors in patients with advanced solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P045.

Checkpoint Inhibition Reduces the Threshold for Drug-Specific T-Cell Priming and Increases the Incidence of Sulfasalazine Hypersensitivity.

An emerging clinical issue associated with immune-oncology agents is the collateral effects on the tolerability of concomitant medications. One report of this phenomenon was the increased incidence of hypersensitivity reactions observed in patients receiving concurrent immune checkpoint inhibitors (ICIs) and sulfasalazine (SLZ). Thus, the aim of this study was to characterize the T cells involved in the pathogenesis of such reactions, and recapitulate the effects of inhibitory checkpoint blockade on de-novo priming responses to compounds within in vitro platforms. A regulatory competent human dendritic cell/T-cell coculture assay was used to model the effects of ICIs on de novo nitroso sulfamethoxazole- and sulfapyridine (SP) (the sulfonamide component of SLZ) hydroxylamine-specific priming responses. The role of T cells in the pathogenesis of the observed reactions was explored in 3 patients through phenotypic characterization of SP/sulfapyridine hydroxylamine (SPHA)-responsive T-cell clones (TCC), and assessment of cross-reactivity and pathways of T-cell activation. Augmentation of the frequency of responding drug-specific T cells and intensity of the T-cell response was observed with PD-1/PD-L1 blockade. Monoclonal populations of SP- and SPHA-responsive T cells were isolated from all 3 patients. A core secretory effector molecule profile (IFN-γ, IL-13, granzyme B, and perforin) was identified for SP and SPHA-responsive TCC, which proceeded through Pi and hapten mechanisms, respectively. Data presented herein provides evidence that drug-responsive T cells are effectors of hypersensitivity reactions observed in oncology patients administered ICIs and SLZ. Perturbation of drug-specific T-cell priming is a plausible explanation for clinical observations of how an increased incidence of these adverse events is occurring.

Multi-Tissue Characterization of GILZ Expression in Dendritic Cell Subsets at Steady State and in Inflammatory Contexts.

Dendritic cells (DCs) are key players in the control of tolerance and immunity. Glucocorticoids (GCs) are known to regulate DC function by promoting their tolerogenic differentiation through the induction of inhibitory ligands, cytokines, and enzymes. The GC-induced effects in DCs were shown to critically depend on increased expression of the Glucocorticoid-Induced Leucine Zipper protein (GILZ). GILZ expression levels were further shown to control antigen-presenting cell function, as well as T-cell priming capacity of DCs. However, the pattern of GILZ expression in DC subsets across tissues remains poorly described, as well as the modulation of its expression levels in different pathological settings. To fill in this knowledge gap, we conducted an exhaustive analysis of GILZ relative expression levels in DC subsets from various tissues using multiparametric flow cytometry. This study was performed at steady state, in the context of acute as well as chronic skin inflammation, and in a model of cancer. Our results show the heterogeneity of GILZ expression among DC subsets as well as the complexity of its modulation, that varies in a cell subset- and context-specific manner. Considering the contribution of GILZ in the control of DC functions and its potential as an immune checkpoint in cancer settings, these results are of high relevance for optimal GILZ targeting in therapeutic strategies.

Three-Dimensional Single Molecule Localization Microscopy Reveals the Topography of the Immunological Synapse at Isotropic Precision below 15 nm

T-cells engage with antigen-presenting cells in search for antigenic peptides and form transient interfaces termed immunological synapses. Synapse topography affects receptor binding rates and the mutual segregation of proteins due to size exclusion effects. It is hence important to determine the 3D topography of the immunological synapse at high precision. Current methods provide only rather coarse images of the protein distribution within the synapse. Here, we applied supercritical angle fluorescence microscopy combined with defocused imaging, which allows three-dimensional single molecule localization microscopy (3D-SMLM) at an isotropic localization precision below 15 nm. Experiments were performed on hybrid synapses between primary T-cells and functionalized glass-supported lipid bilayers. We used 3D-SMLM to quantify the cleft size within the synapse by mapping the position of the T-cell receptor (TCR) with respect to the supported lipid bilayer, yielding average distances of 18 nm up to 31 nm for activating and nonactivating bilayers, respectively.

Imaging of Primary and Metastatic Tumors Treated With Radiotherapy-Directed Antigen Capturing Nanoparticles, Dissociation of Circulating Tumor Cell (CTC)-Clusters, and Reduction of CTC Extravasation Under PDL-1 Blockade

<h3>Purpose/Objective(s)</h3> We investigated the efficacy of a combination of radiotherapy with antigen-capturing nanoparticles (AC-NPs; 123 ± 45 nm) containing digitoxin and anti-PODXL siRNA encapsulated in nanocapsules (567 ± 38 nm) that release their contents upon radiation exposure. We performed two radiotherapy sessions. Treatment with AC-NPs achieved successful immuno-radiotherapeutic effects on the primary tumors. An abscopal effect was also observed in the metastatic tumors by dendritic cell (DC)-mediated T-cell priming with PD-L1 blockade. New metastasis reduction was achieved by the dissociation of circulating tumor cell (CTC)-clusters by digitoxin and by reducing the extravasation of the dissociated CTCs via siRNA-mediated silencing of PODXL. <h3>Materials/Methods</h3> For session 1; nanocapsules were generated by mixing iopamiron and 400 μg anti-PD-L1 antibody (Ab) with a 1.0 mL solution containing 4.0% alginate, 3.0% hyaluronate, and 1 µg/mL P-selectin. This mixture was sprayed into 0.5 mmol/l FeCl₂ supplemented with 1 µg/mL anti-VEGFR-1/2 Ab. For session two, 400 nM digitoxin and 50 nM anti-PODXL siRNA were encapsulated in poly lactic-co-glycolic acid (PLGA) AC-NPs using the nanoprecipitation method. The particles were mixed with the above cocktail and sprayed into 0.5 mM/L FeCl₂ with 1 µg/mL anti-P-selectin Ab. This yielded encapsulated PLGA AC-NPs containing digitoxin. In session 1; 1 × 10¹⁰ nanocapsules were intravenously injected in BALB/c mice exhibiting primary 4TI mammary carcinoma in the left hind leg and lung metastases. Tumor accumulation was monitored using CT imaging. Subsequently, 10/20 Gy ⁶⁰Co γ-radiation was locally administered to the primary tumors. In session 2; 1 × 10¹⁰ nanocapsules were intravenously injected and allowed to interact with P-selectin for 9 h; 10/20 Gy ⁶⁰Co γ-radiation was locally administered. <h3>Results</h3> In session 1, CT revealed the accumulation of anti-VEGFR-1/2 nanocapsules around the primary and metastatic tumors. The microcapsules released P-selectin and anti-PD-L1 Ab in response to the initial irradiation. After session 2, the nanocapsules amassed around the primary tumor via a P-selectin Ag-Ab reaction. PLGA AC-NPs captured tumor-derived protein antigens released by the second radiation dose and transported them to DCs. This intensified DC-associated cross-priming of CD8⁺ T-cells. Primed CD8⁺ T-cells attacked PD-L1 and suppressed primary and metastatic tumors. PLGA AC-NPs also released digitoxin and anti-PODXL siRNA. The released digitoxin dissociated CTC clusters into single CTCs, and extravasation was reduced by siRNA-mediated silencing of PODXL, which reduced new metastasis formation. These treatments significantly increased the antitumor effect (EF 1.5) and reduced metastasis by 87.2%. <h3>Conclusion</h3> CT-detectable microcapsules exhibited targeted AC-NP-mediated immunotherapeutic benefits. They caused abscopal effects and reduced new metastasis formation, which improved the diagnosis and treatment of the tumor.

ACKR4 in Tumor Cells Regulates Dendritic Cell Migration to Tumor-Draining Lymph Nodes and T-Cell Priming.

Simple SummaryOur study demonstrated that Atypical Chemokine Receptor 4 (ACKR4) was downregulated in human colorectal cancer (CRC) compared with normal colon tissues. Loss of ACKR4 in human CRC was associated with a weak anti-tumor immune response. Knockdown of ACKR4 in tumor cells impairs the dendritic cell migration from the tumor to the tumor-draining lymph nodes (TdLNs), causing inadequate tumor-specific T-cell expansion and insensitivity to immune checkpoint blockades. However, loss of ACKR4 in stromal cells does not significantly affect anti-tumor immunity. In human CRC, high expression of microRNA-552 was a mechanism leading to ACKR4 downregulation. Our study revealed a novel mechanism that leads to the poor immune response in a subset of CRC and will contribute to the framework for identifying new therapies against this deadly cancer.Colorectal cancer (CRC) is one of the most common malignancies in both morbidity and mortality. Immune checkpoint blockade (ICB) treatments have been successful in a portion of mismatch repair-deficient (dMMR) CRC patients but have failed in mismatch repair-proficient (pMMR) CRC patients. Atypical Chemokine Receptor 4 (ACKR4) is implicated in regulating dendritic cell (DC) migration. However, the roles of ACKR4 in CRC development and anti-tumor immunoregulation are not known. By analyzing human CRC tissues, transgenic animals, and genetically modified CRC cells lines, our study revealed an important function of ACKR4 in maintaining CRC immune response. Loss of ACKR4 in CRC is associated with poor immune infiltration in the tumor microenvironment. More importantly, loss of ACKR4 in CRC tumor cells, rather than stromal cells, restrains the DC migration and antigen presentation to the tumor-draining lymph nodes (TdLNs). Moreover, tumors with ACKR4 knockdown become less sensitive to immune checkpoint blockade. Finally, we identified that microRNA miR-552 negatively regulates ACKR4 expression in human CRC. Taken together, our studies identified a novel and crucial mechanism for the maintenance of the DC-mediated T-cell priming in the TdLNs. These new findings demonstrate a novel mechanism leading to immunosuppression and ICB treatment resistance in CRC.

Immune landscape in vulvar cancer-draining lymph nodes indicates distinct immune escape mechanisms in support of metastatic spread and growth

BackgroundTherapeutic immune intervention is highly dependent on the T-cell priming and boosting capacity of tumor-draining lymph nodes (TDLN). In vulvar cancer, in-depth studies on the immune status of (pre)metastatic TDLN...

OAB-033: Loss-of-function of GABARAP drives tumor resistance to bortezomib-induced immunogenic cell death in multiple myeloma

<h3>Background</h3> Resistance to immune approaches poses a major challenge to effective immunotherapy and long-term clinical outcome in multiple myeloma (MM). Here we identified loss-of-function of gamma-aminobutyric acid receptor-associated protein (GABARAP) as a tumor-intrinsic mechanism of resistance to bortezomib (BTZ)-induced immunogenic cell death (ICD), the immunogenic consequence of apoptosis resulting in specific anti-MM immunity via T-cell priming by dendritic cells (DCs). <h3>Methods</h3> We found that BTZ induces ICD in human and murine MM cell lines that is dependent on the exposure of calreticulin (CALR), which drives DCs-mediated phagocytosis of MM cells. DCs-phagocytosis is followed by a specific T cell activation, with a significant increase of CD4+ effector memory (EM), total CD8+, CD8+ EM, and CD8+ terminally differentiated EM cells. Isolated T cells after co-cultures showed the presence of MM specific CTLs that were able to efficiently induce lysis of MM cells. Our results were validated using primary cells from MM patients. Moreover, induction of a protective immune response was confirmed in vivo. Specifically, treatment of 5TGM1 tumors with BTZ induced a tumor regression in a syngeneic model; and injection of live 5TGM1WT two weeks after regression did not result in tumor development, consistent with induction of immunological memory as confirmed by ELISPOT of mouse splenocytes. We identified a specific ICD signature induced by BTZ in mice; and we found that increased expression of the human orthologs of this signature was positively correlated with OS (p=0.01) in patients enrolled in the IFM/DFCI 2009 study. Notably, these functional immunologic sequelae were abrogated after BTZ treatment of CALRKO MM cells both in vitro and in vivo, confirming the obligate role of CALR exposure in the ICD process. <h3>Results</h3> By interrogating the IFM/DFCI dataset and focusing on genes involved in ICD processes that were correlated with MM patients clinical outcome, we found that low levels of GABARAP (chr17p13.1), an autophagy regulator and putative CALR binding partner, negatively impact MM patient clinical outcome (EFS, p=0.0032); even excluding HR patients with 17p deletion (EFS, p=0.018). Interestingly, KMS11 cells carrying monoallelic deletion of GABARAP were resistant to induction of ICD by BTZ; and sensitivity was restored after overexpression of the gene. Moreover, GABARAPKO in 3 ICD-sensitive cell lines abrogated the induction of ICD by BTZ; and add-back experiments by pre-treatment with recombinant CALR or GABARAP overexpression in KO clones restored ICD. Finally, CyTOF confirmed that treatment of GABARAPKO cells with BTZ failed to activate an efficient T cell response. <h3>Conclusions</h3> our study demonstrates that loss-of-function of GABARAP, particularly in HR patients with 17p deletion, contributes to tumor immune evasion and ICD resistance. These studies provide the framework for novel combination treatments to restore anti-MM immunity and improve patient outcome in HR MM.

Prec-O1-03 - Understanding the function of CD30 in cutaneous T-cell lymphoma: implications for therapy and prognosis

<h3>Introduction and objectives:</h3> Targeted therapies for cutaneous T-cell lymphoma (CTCL) are limited and curative approaches are still lacking. Relapses and drug-induced side effects are major challenges in the therapeutic management of CTCL patients, resulting in an urgent need for new, effective therapies. Expression of CD30 receptor is usually restricted to a small population of activated B- and T-cells, but is also found in hematologic malignancies, such as CTCL. In mycosis fungoides (MF) its expression seems to be associated with advanced disease stage and large-cell transformation. However, the functional role of CD30 in CTCL remains unknown. Consequently, this study aims to determine 1) CD30 and CD30 ligand (CD30L) expression in CTCL cell lines and primary CD4-positive cells from CTCL patients, 2) the dynamics of CD30 expression over time and after treatment, 3) CD30 function <i>in vitro</i>, <i>ex vivo</i> and <i>in vivo</i>, 4) mechanisms involved in CD30 signaling. <h3>Materials and methods:</h3> The CTCL cell lines HH, SeAx and HUT-78 were used for <i>in vitro</i> experiments. For <i>ex vivo</i> analysis, primary CD4-positive T-cells isolated from fresh blood samples of patients with Sézary syndrome were used. Cells were challenged with dimethyl fumarate for 24 hours. CD30 was stimulated via its natural ligand. Methods used for expression and pathway analysis were FACS, western blot, PCR and luciferase assay. For <i>in vivo</i> experiments, CTCL cells were suspended in PBS/Matrigel (1:1, vol/vol) and injected intradermally into the right flank of female NOD/SCID/Il2Rg null (NSG) mice. After development of tumors, CD30L was injected into the lesions. <h3>Results:</h3> We found that CD30 activation can influence viability of CTCL cells <i>in vitro</i>, which is currently under investigation in an <i>in vivo</i> setting. Involved mechanisms include the MEK/ERK and NF-κB pathway. The highest CD30 expression was detected in the HH cell line. The primary CD4-positive cells analyzed so far were negative for CD30 surface expression. However, intracellular CD30 protein was detectable via western blot. CD30 levels were influenced by cell culture conditions in HH cells and significantly reduced upon therapeutic NFκB inhibition in HH, SeAx and HUT-78 cells. <h3>Conclusions:</h3> Our results indicate a therapeutic potential of CD30 receptor stimulation as a new targeted therapy in CD30-positive MF <i>in vitro</i>, which has to be proven <i>in vivo</i>. Furthermore, we found a dynamic nature of CD30 expression in CTCL, which might account for the described effectiveness of the antibody-drug conjugate brentuximab vedotin directed to CD30 also in patients diagnosed as CD30 low-expressing.

In This Issue

In This Issue

Low neoantigen expression and poor T-cell priming underlie early immune escape in colorectal cancer.

Immune evasion is a hallmark of cancer, and therapies that restore immune surveillance have proven highly effective in cancers with high tumor mutation burden (TMB) (e.g., those with microsatellite instability (MSI)). Whether low TMB cancers, which are largely refractory to immunotherapy, harbor potentially immunogenic neoantigens remains unclear. Here, we show that tumors from all patients with microsatellite stable (MSS) colorectal cancer (CRC) express clonal predicted neoantigens despite low TMB. Unexpectedly, these neoantigens are broadly expressed at lower levels compared to those in MSI CRC. Using a versatile platform for modulating neoantigen expression in CRC organoids and transplantation into the distal colon of mice, we show that low expression precludes productive cross priming and drives immediate T cell dysfunction. Strikingly, experimental or therapeutic rescue of priming rendered T cells capable of controlling tumors with low neoantigen expression. These findings underscore a critical role of neoantigen expression level in immune evasion and therapy response.

Phagocytes produce prostaglandin E2 in response to cytosolic Listeria monocytogenes.

Listeria monocytogenes is an intracellular bacterium that elicits robust CD8+ T-cell responses. Despite the ongoing development of L. monocytogenes-based platforms as cancer vaccines, our understanding of how L. monocytogenes drives robust CD8+ T-cell responses remains incomplete. One overarching hypothesis is that activation of cytosolic innate pathways is critical for immunity, as strains of L. monocytogenes that are unable to access the cytosol fail to elicit robust CD8+ T-cell responses and in fact inhibit optimal T-cell priming. Counterintuitively, however, activation of known cytosolic pathways, such as the inflammasome and type I IFN, lead to impaired immunity. Conversely, production of prostaglandin E2 (PGE2) downstream of cyclooxygenase-2 (COX-2) is essential for optimal L. monocytogenes T-cell priming. Here, we demonstrate that vacuole-constrained L. monocytogenes elicit reduced PGE2 production compared to wild-type strains in macrophages and dendritic cells ex vivo. In vivo, infection with wild-type L. monocytogenes leads to 10-fold increases in PGE2 production early during infection whereas vacuole-constrained strains fail to induce PGE2 over mock-immunized controls. Mice deficient in COX-2 specifically in Lyz2+ or CD11c+ cells produce less PGE2, suggesting these cell subsets contribute to PGE2 levels in vivo, while depletion of phagocytes with clodronate abolishes PGE2 production completely. Taken together, this work demonstrates that optimal PGE2 production by phagocytes depends on L. monocytogenes access to the cytosol, suggesting that one reason cytosolic access is required to prime CD8+ T-cell responses may be to facilitate production of PGE2.