Enhanced Antigen Presentation Research Articles

Self-Tolerance of Vascular Tissues Is Broken Down by Vascular Dendritic Cells in Response to Systemic Inflammation to Initiate Regional Autoinflammation.

The correlation of infections with vascular autoinflammatory diseases such as vasculitis and atherosclerosis has been long recognized, and progressive inflammation with the formation of tertiary lymphoid organs in arterial adventitia intensively studied, the immunological basis of the nondiseased vasculatures that predispose to subsequent vascular autoimmunity during inflammation, however, is not well characterized. Here, we investigated the vascular immunity in situ of steady-state C57BL/6 mice and found that healthy vascular tissues contained a comprehensive set of immune cells with relatively higher proportion of innate components than lymphoid organs. Notably, a complete set of dendritic cell (DC) subsets was observed with monocyte-derived DCs (moDCs) constituting a major proportion; this is in contrast to moDCs being considered rare in the steady state. Interestingly, these vascular DCs constitutively expressed more suppressive factors with cDC1 for PD-L1 and moDCs for IL-10; this is concordant with the inhibitive phenotype of T cells in normal vascular tissues. The immunotolerant state of the vascular tissues, however, was readily eroded by systemic inflammation, demonstrated by the upregulation of proinflammatory cytokines and enhanced antigen presentation by vascular DCs to activate both cellular and humoral immunity in situ, which ultimately led to vascular destruction. Different vascular DC subsets elicited selective effects: moDCs were potent cytokine producers and B-cell activators, whereas cDCs, particularly, cDC1, were efficient at presenting antigens to stimulate T cells. Together, we unveil regional immunological features of vascular tissues to explain their dual facets under physiological versus pathological conditions for the better understanding and treatment of cardiovascular autoinflammation.

Impact of Antigen Presentation Mechanisms on Immune Response in Autoimmune Hepatitis.

The liver is a very tolerogenic organ. It is continually exposed to a multitude of antigens and is able to promote an effective immune response against pathogens and simultaneously immune tolerance against self-antigens. In spite of strong peripheral and central tolerogenic mechanisms, loss of tolerance can occur in autoimmune liver diseases, such as autoimmune hepatitis (AIH) through a combination of genetic predisposition, environmental factors, and an imbalance in immunological regulatory mechanisms. The liver hosts several types of conventional resident antigen presenting cells (APCs) such as dendritic cells, B cells and macrophages (Kupffer cells), and unconventional APCs including liver sinusoidal endothelial cells, hepatic stellate cells and hepatocytes. By standard (direct presentation and cross-presentation) and alternative mechanisms (cross-dressing and MHC class II-dressing), liver APCs presents self-antigen to naive T cells in the presence of costimulation leading to an altered immune response that results in liver injury and inflammation. Additionally, the transport of antigens and antigen:MHC complexes by trogocytosis and extracellular vesicles between different cells in the liver contributes to enhance antigen presentation and amplify autoimmune response. Here, we focus on the impact of antigen presentation on the immune response in the liver and on the functional role of the immune cells in the induction of liver inflammation. A better understanding of these key pathogenic aspects could facilitate the establishment of novel therapeutic strategies in AIH.

Photoimmunotherapy: A New Paradigm in Solid Tumor Immunotherapy

In recent years, the incidence of cancer has been increasing worldwide. Conventional cancer treatments include surgery, chemotherapy, and radiation, which mostly kill tumor cells at the expense of normal and immune cells. Although immunotherapy is an accurate, rapid, efficient tumor immune treatment, it causes serious adverse reactions, such as cytokine release syndrome (CRS) and neurotoxicity. Therefore, there is an urgent need to develop an effective and nontoxic procedure for immunotherapy. The clinical combination of phototherapy and immunoadjuvant therapy can induce immunogenic cell death and enhance antigen presentation synergy. It also causes a systemic antitumor immune response to manage residual tumors and distant metastases. Photoimmunotherapy (PIT) is a tumor treatment combining phototherapy with immunotherapy based on injecting a conjugate photosensitizer (IR700) and a monoclonal antibody (mAb) to target an expressed antigen on the tumor surface. This combination can enhance the immune response ability, thus having a good effect on the treatment of residual tumor and metastatic cancer. In this review, we summarize the recent progress in photoimmunotherapy, including photoimmunoconjugate (PIC), the activation mechanism of immunogenic cell death (ICD), the combination therapy model, opportunities and prospects. Specifically, we aim to provide a promising clinical therapy for solid tumor clinical transformation.

Dendritic Cell-Based Immunotherapies and their Potential use in Colorectal Cancer Immunotherapy.

Dendritic cells (DCs) are professional antigen-presenting cells, which are resident or proliferating in organs. Major histocompatibility complex (MHC) Class I and II on DCs in normal steady conditions process and present antigens including cancer antigens. Many approaches are used to enhance antigen presentation process of DCs and capture cancer cells. DCs are harvested from cancer patients and manipulated ex vivo in DC-based cancer immunotherapy. In addition, DCs' vaccines and other anticancer therapy combinations were discussed to optimize DCs' efficiency for cancer immunotherapy. This review addressed the use of the human conventional type-1 DCs, OX40+ plasmacytoid DCs, and DCs-derived exosomes. In addition, different combinations with DCs therapy such as combination with the monoclonal antibody, cytokine-induced killer cells, adjuvants, chemotherapy (DCs-based chemoimmunotherapy), and nanoparticles were listed and explored for their effectiveness against cancer, and mainly against colorectal cancer.

N6-methyladenosine Regulator-Mediated Immune Genes Identify Breast Cancer Immune Subtypes and Predict Immunotherapy Efficacy.

Breast cancer (BRCA) is a heterogeneous malignancy closely related to the tumor microenvironment (TME) cell infiltration. N6-methyladenosine (m6A) modification of mRNA plays a crucial regulator in regulating the immune microenvironment of BRCA. Immunotherapy represents a paradigm shift in BRCA treatment; however, lack of an appropriate approach for treatment evaluation is a significant issue in this field. In this study, we attempted to establish a prognostic signature of BRCA based on m6A-related immune genes and to investigate the potential association between prognosis and immunotherapy. We comprehensively evaluated the m6A modification patterns of BRCA tissues and non-tumor tissues from The Cancer Genome Atlas and the modification patterns with TME cell-infiltrating characteristics. Overall, 1,977 TME-related genes were identified in the literature. Based on LASSO and Cox regression analyses, the m6A-related immune score (m6A-IS) was established to characterize the TME of BRCA and predict prognosis and efficacy associated with immunotherapy. We developed an m6A-IS to effectively predict immune infiltration and the prognosis of patients with BRCA. The prognostic score model represented robust predictive performance in both the training and validation cohorts. The low-m6A-IS group was characterized by enhanced antigen presentation and improved immune checkpoint expression, further indicating sensitivity to immunotherapy. Compared with the patients in the high-score group, the overall survival rate after treatment in the low-score group was significantly higher in the testing and validation cohorts. We constructed an m6A-IS system to examine the ability of the m6A signature to predict the infiltration of immune cells of the TME in BRCA, and the m6A-IS system acted as an independent prognostic biomarker that predicts the response of patients with BRCA in immunotherapy.

Abstract P111: Therapeutic KRASG12C inhibition alleviates KRAS-driven immunosuppression

Abstract Therapeutic KRASG12C inhibitors may substantially improve KRAS-mutant cancer patient care, considering the encouraging clinical data emerging from ongoing clinical trials. Since oncogene-targeting therapies often lead to resistance, the development of combinatorial therapies will be of utmost importance to achieve long-lasting tumour control. Given the known immunosuppressive role of KRAS, clinical trials investigating the combination of KRASG12C inhibitors with immune checkpoint blockade are currently underway for NSCLC patients. Here, we show that KRASG12C inhibition using MRTX1257 alleviates KRAS-driven immune suppression, through a myriad of mechanisms including the augmentation of tumour cell-intrinsic interferon responses. Despite harbouring a large number of somatic mutations, orthotopic 3LL (KRASG12CDNRAS) tumours are able to evade anti-tumour immune responses. Combining multicolour flow cytometry, RNA sequencing and imaging mass cytometry, we observed significantly increased T cell activation and infiltration after MRTX1257 treatment in addition to enhanced antigen presentation cells and gene sets, and reduced myeloid cells, creating an immune permissive microenvironment in these lung tumours. Despite these profound changes, combination of KRASG12C inhibitors and anti-PD-(L)1 antibodies fail to show synergism in our model, in contrast with Amgen’s published data using an immunogenic model of colorectal cancer. This data suggests that a pre-existing anti-tumour immunity may be crucial for clinical responses to KRAS inhibitors and immune checkpoint blockade combinations. Henceforth, ongoing clinical trials investigating this approach will likely only benefit a subset of patients and further research will be needed to examine novel therapeutic approaches and combinations for refractory patients. Citation Format: Edurne Mugarza, Febe Van Maldegem, Jesse Boumelha, Chris Moore, Sareena Rana, Miriam Llorian Sopena, Phil East, Miriam Molina Arcas, Julian Downward. Therapeutic KRASG12C inhibition alleviates KRAS-driven immunosuppression [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 P111.

164P Preliminary results from a phase I/II study of BDC-1001, a novel HER2 targeting TLR7/8 immune-stimulating antibody conjugate (ISAC), in patients (pts) with advanced HER2-expressing solid tumors

BDC-1001 is a novel ISAC consisting of an investigational trastuzumab biosimilar chemically conjugated to a TLR7/8 agonist with a non-cleavable linker. BDC-1001 elicits myeloid activation and enhances antigen presentation leading to antibody-mediated effector functions that promote T-cell activation and a durable adaptive immune response.

Combination of epigenetic regulation with gene therapy-mediated immune checkpoint blockade induces anti-tumour effects and immune response in vivo

Immunotherapy has become a powerful cancer treatment, but only a small fraction of patients have achieved durable benefits due to the immune escape mechanism. In this study, epigenetic regulation is combined with gene therapy-mediated immune checkpoint blockade to relieve this immune escape mechanism. PPD (i.e., mPEG-b-PLG/PEI-RT3/DNA) is developed to mediate plasmid-encoding shPD-L1 delivery by introducing multiple interactions (i.e., electrostatic, hydrogen bonding, and hydrophobic interactions) and polyproline II (PPII)-helix conformation, which downregulates PD-L1 expression on tumour cells to relieve the immunosuppression of T cells. Zebularine (abbreviated as Zeb), a DNA methyltransferase inhibitor (DNMTi), is used for the epigenetic regulation of the tumour immune microenvironment, thus inducing DC maturation and MHC I molecule expression to enhance antigen presentation. PPD plus Zeb combination therapy initiates a systemic anti-tumour immune response and effectively prevents tumour relapse and metastasis by generating durable immune memory. This strategy provides a scheme for tumour treatment and the inhibition of relapse and metastasis.

Pembrolizumab Added to First-Line Cyclophosphamide, Bortezomib and Dexamethasone in Newly Diagnosed Transplant Non-Eligible Myeloma Patients: A Phase 2A Open Label, Multi Centre Study

Background: Despite recent improvements, myeloma is still incurable. There is need to add new therapeutic tools. The combination of the proteasome inhibitor (PI) Bortezomib, an alkylator (Cyclophosphamide or Melphalan) and Dexamethasone (Dex) /Prednisone is a widely used first-line therapy. Most patients achieve a rapid response within the first two cycles. The lack of a deep response to the bortezomib-based therapy predicts an inferior outcome, a poor response to newer PIs and limited therapeutic options. Different studies highlighted the role of the PD1/PDL1 pathway in myeloma immune escape and progression. The expression of PDL1 by myeloma cells is higher in relapsed or refractory disease and on minimal residual disease cells. At least one preclinical study showed that the binding of PD1 to PDL1 expressed by the myeloma cells leads to a reverse signal that induces resistance to bortezomib and melphalan. Also, Bortezomib is known to enhance anti-myeloma immune response by different mechanisms, including stimulating myeloma cell apoptosis, sensitizing myeloma cells to natural killer cell mediated killing and enhancing antigen presentation by dendritic cells. This suggests a mutual potentiation between Pembrolizumab and Cyclophosphamide, Bortezomib and Dex (CyBorD). An early immune-based intervention is likely more beneficial. With time and disease progression, the host is more immune-compromised and the disease is more refractory with redundant escape mechanisms. The benefits of early intervention are carefully weighed against the potential risks and other available therapeutic options. The adaptive design of this CMRG-006 trial is meant to select the population with the highest need for treatment improvement while still in the early phase of therapy. We expect the combination of Pembrolizumab and CyBorD to be more effective and well tolerated.Design and Methods: Phase 2A, pilot study. Non-transplant eligible newly diagnosed myeloma patients (NTE-NDMM) will start their standard nine cycles of CyBorD (Cyclophosphamide 300 mg/m 2 PO, Bortezomib 1.5 mg/m 2 SC, and Dex 40 mg PO, all given on days 1, 8, 15 and 22 of 28-day cycle). Patients not having primary disease progression and achieving less than Very Good Partial Response (VGPR) after 2 cycles will be screened for this study during cycle 3. Eligible patients will receive Pembrolizumab 200 mg IV every 3 weeks starting with day 1 of cycle 4 (P-CyBorD). After completion of cycle nine of CyBorD, Pembrolizumab will be administered as a single agent for up to total of 35 doses. The objectives are to evaluate the efficacy and tolerability of P-CyBorD. The key inclusion criteria are having a NTE-NDMM with a measurable disease; no primary progressive disease and not achieving VGPR after two cycles of CyBorD; adequate performance status; and adequate organ functions. The key exclusion criteria include previous exposure to anti-PD1 therapy, intolerance to CyBorD, adverse cardiac history, pulmonary disease, central nervous system disease, congenital or acquired immune suppression, and AL amyloidosis. S ample Size: A maximum of 20 patients will be enrolled to ensure 16 or more patients evaluable for response. Patients with less than VGPR after two cycles of CyBorD have ≤20% probability to achieve ≥VGPR after cycle 9. With the addition of Pembrolizumab, a minimal clinically important difference (MCID) of 10% would be meaningful, and a 50% response rate (≥VGPR) in 16 evaluable patients will be statistically significant.This study is currently recruiting. Clinicaltrials.gov #: NCT04258683. DisclosuresKotb: Amgen: Honoraria; BMS: Honoraria; Merck: Honoraria, Research Funding; Karyopharm: Current holder of individual stocks in a privately-held company; Takeda: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Sanofi: Honoraria, Research Funding; Akcea: Honoraria; Pfizer: Honoraria. Othman: Takeda: Honoraria; Roche: Honoraria; Novartis: Honoraria; Janssen: Honoraria; Sanofi: Honoraria; Celgene: Honoraria. Reece: Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Sanofi: Honoraria; Millennium: Research Funding; BMS: Honoraria, Research Funding; Karyopharm: Consultancy, Research Funding; Amgen: Consultancy, Honoraria; GSK: Honoraria.

Hematopoietic Stem Cells Undergo Immune Training and Constitute a Long-Term Reservoir for Hyper-Inflammatory Macrophages in a Mouse Model of Chronic Autoimmunity

Auto-immune diseases (AD) are characterized by repeated flares of disease activity separated by periods of remission. Cycles of AD remission and relapse can occur even with therapeutic intervention and contribute to AD morbidity. Paradoxically, during remission myeloid cells retain increased expression of genes related to interferon signaling and antigen presentation. Given the relatively short lifespan of myeloid cells, these observations imply the existence of a clonal reservoir fueling AD relapse. Recent literature describes hematopoietic stem cells (HSC) as a cellular source for trained myeloid cells in response to immune stimuli. Thus, we hypothesize that HSC may also retain a long-term memory of chronic autoimmune inflammation, thereby providing a continuous supply of myeloid cells that promote AD pathogenesis.To test this hypothesis, we have combined high-throughput molecular and cytokine profiling approaches with functional assays to address heritable changes in immune function using the pristane-induced mouse model of systemic lupus erythematosus (SLE). Eight weeks after pristane injection, we observe significant myeloid lineage expansion in the bone marrow (BM), including mature myeloid cells, granulocyte/monocyte progenitors (GMP) and multipotent progenitor (MPP) populations. To understand the impact of SLE-like disease on the molecular programming of the hematopoietic system, we performed RNA-seq analyses of BM Mon and HSC. As anticipated, BM Mon activated inflammatory programs and antigen presentation genes, which overlapped with gene signatures of human monocytes from SLE patients. HSC also activated innate defense gene programs resembling signatures of trained immunity, thus establishing the potential for autoimmune inflammation to induce immune training in HSC.To establish whether these molecular programs potentiate myeloid cell function, we generated BM-derived macrophages (BMDM) from control and pristane-induced mice. BMDMs from pristane-induced mice exhibited significantly increased capacity to kill Mycobacterium avium. Further, co-culture of T cells with BMDMs from pristane-induced mice significantly boosted T cell proliferation, indicative of enhanced antigen presentation. To establish whether HSC from pristane-induced mice propagate molecular memory of SLE-like disease to myeloid progeny, we transplanted stringently enriched (LSK/SLAM/CD34 -/EPCR +) long-term (LT)-HSC into lethally irradiated recipient mice. 18 weeks post transplant, mice transplanted with LT-HSC from pristane-induced donors had a small but significant reduction in donor BM HSC chimerism but did not exhibit overt changes in lineage output. Strikingly, BMDMs from pristane-induced donors showed increased bacterial killing and inflammatory cytokine generation following M. avium challenge, as well as increased capacity to induce antigen-specific T cell proliferation. Thus, LT-HSC retain and pass on altered functional properties to myeloid cells, even in the absence of AD activity.To characterize the molecular mechanisms underlying HSC-propagated alterations in myeloid cell function, we ran RNA-seq on donor-derived GMP and BMDMs. Notably, GMP derived from pristane-induced donor LT-HSC had increased expression of Fos and Jun/b/d, key molecular drivers of immune training in stem cells. Likewise, pristane-induced donor BMDMs maintained increased expression of IFN-regulated genes including MHC-I, a gene also overexpressed in PBMCs from human SLE patients. Consistent with these findings, re-stimulation of recipient mice with pristane led to a significant increase in cytokine-producing lymph node T cells versus recipient mice transplanted with control HSC, further supporting a model of immune training.Overall, these data show that chronic autoimmune inflammation can induce in HSC a heritable trained immunity phenotype that is transmitted to myeloid progeny, enhancing their functional activity. Ongoing studies are establishing the capacity for this phenotype to exacerbate AD. We are also testing the importance of molecular players identified above in establishing AD-related immune training and assessing the potential for therapeutic interventions to disrupt HSC memory in this setting. Our data thus stand to establish a new paradigm for trained immunity in HSC as a key contributor to AD pathology and relapse. DisclosuresNo relevant conflicts of interest to declare.

Pembrolizumab Plus Vorinostat Induces Responses in Patients with Hodgkin Lymphoma Who Are Refractory to Prior PD-1 Blockade

Pembrolizumab Plus Vorinostat Induces Responses in Patients with Hodgkin Lymphoma Who Are Refractory to Prior PD-1 Blockade

Review: Development of SARS-CoV-2 immuno-enhanced COVID-19 vaccines with nano-platform.

Vaccination is the most effective way to prevent coronavirus disease 2019 (COVID-19). Vaccine development approaches consist of viral vector vaccines, DNA vaccine, RNA vaccine, live attenuated virus, and recombinant proteins, which elicit a specific immune response. The use of nanoparticles displaying antigen is one of the alternative approaches to conventional vaccines. This is due to the fact that nano-based vaccines are stable, able to target, form images, and offer an opportunity to enhance the immune responses. The diameters of ultrafine nanoparticles are in the range of 1–100 nm. The application of nanotechnology on vaccine design provides precise fabrication of nanomaterials with desirable properties and ability to eliminate undesirable features. To be successful, nanomaterials must be uptaken into the cell, especially into the target and able to modulate cellular functions at the subcellular levels. The advantages of nano-based vaccines are the ability to protect a cargo such as RNA, DNA, protein, or synthesis substance and have enhanced stability in a broad range of pH, ambient temperatures, and humidity for long-term storage. Moreover, nano-based vaccines can be engineered to overcome biological barriers such as nonspecific distribution in order to elicit functions in antigen presenting cells. In this review, we will summarize on the developing COVID-19 vaccine strategies and how the nanotechnology can enhance antigen presentation and strong immunogenicity using advanced technology in nanocarrier to deliver antigens. The discussion about their safe, effective, and affordable vaccines to immunize against COVID-19 will be highlighted.

Focused ultrasound immunomodulation on mononuclear phagocytes in the brain

Focused ultrasound (FUS)-mediated blood–brain barrier opening is an attractive method of treating neurological diseases, such as glioblastoma (GBM) and Alzheimer’s disease (AD). As an established drug delivery approach, FUS has also been associated with the stimulation of neuro-inflammation, including the morphological activation of resident microglia. Here we report FUS immunomodulation effects may also include the enhanced recruitment and activation of other mononuclear phagocytes in murine models of GBM (GL261) and AD (APP/PS1dE9) in tests with or without monoclonal antibody-based passive immunotherapy. Immunoprofiling via flow cytometry and immunofluorescent staining were performed in the harvested brains. In the GBM model, our results demonstrated that FUS enhanced antigen presentation behaviors of tumor-associated macrophages without affecting the microglia. FUS also reprogrammed the macrophages locally towards the anti-cancer phenotype. In the AD model, plaque-associated Ly6G+ phagocytes were only present in FUS-treated areas, while microglia activation was found in both antibody-treated groups. Taken together, our results offer new evidence in FUS immunomodulation on the myeloid compartment of the brains in GBM and AD-like mouse models.

Host defense peptides as immunomodulators: The other side of the coin

Host defense peptides (HDPs) exhibit a broad range of antimicrobial and immunomodulatory activities. In this sense, both functions are like different sides of the same coin. The direct antimicrobial side was discovered first, and widely studied for the development of anti-infective therapies. In contrast, the immunomodulatory side was recognized later and in the last 20 years the interest in this field has been continuously growing. Different to their antimicrobial activities, the immunomodulatory activities of host defense peptides are more effective in vivo. They offer a great opportunity for new therapeutic applications in the fields of anti-infective therapy, chronic inflammatory diseases treatment, novel vaccine adjuvants development and anticancer immunotherapy. These immune related functions of HDPs includes chemoattraction of leukocytes, modulation of inflammation, enhancement of antigen presentation and polarization of adaptive immune responses. Our attempt with this review is to make a careful evaluation of different aspects of the less explored, but attractive immunomodulatory side of the HDP functional coin.

Multistage Cooperative Nanodrug Combined with PD-L1 for Enhancing Antitumor Chemoimmunotherapy.

Combinatorial CpG oligonucleotide (CPG) and chemotherapy drug represent a promising approach to reactivate immune system. However, these two agents possess different physicochemical properties, hindering the application of direct self-assembly of these two cargos into a single nanostructure. Here, a multistage cooperative nanodrug is developed by the direct self-assembly of cis-platinum (CDDP, Pt), l-arginine (l-Arg, R), and CPG (defined as PtR/CPG) for antitumor chemoimmunotherapy. First, the CDDP can induce cell apoptosis. Meanwhile, CDDP also promotes the production of H2 O2 , catalyzing the conversion of l-Arg into nitric oxide (NO). The generated NO decreases the multidrug resistance of cells toward CDDP. Thus, the synergistic effects of CDDP and NO can trigger immunogenic cell death to produce tumor-associated antigens (TAAs). The TAAs and CPG will induce the maturation of dendritic cells (DCs) and enhance antigen presentation ability of DCs. In this way, the PtR/CPG can reverse the immunosuppressive microenvironment, sensitizing tumors to immune checkpoint inhibitors mediated by the programmed death-ligand 1 (PD-L1) antibody. Furthermore, the PtR/CPG combined with the PD-L1 antibody decreases the exhaustion and dysfunction of cytotoxic T lymphocytes to elicit durable systemic immune response. As a result, the prepared PtR/CPG nanodrug in combination with PD-L1 may be highly significant for cancer immunotherapy.

Abstract 2755: Single-cell RNA sequencing reveals priming professional antigen-presenting macrophages and chemokine expressing T cells in tumor microenvironment by AXL inhibitor, SKI-G-801

Abstract The mechanistic investigations have defined that AXL inhibition reprograms the immunological microenvironment leading to increased T cells and antigen-presenting cells. However, previous reports did not clearly demonstrate the linchpin mechanism of anti-tumor effects with AXL inhibitors. It may be possible to elucidate the immunological mechanism of AXL inhibitor based on a comprehensive analysis of cell type-specific transcriptomic changes using scRNA-seq. The human CD34-NSG mice were engrafted LUSC tumor. These mice were divided into four dose groups: Vehicle, SKI-G-801, pembrolizumab, and combination groups. The tumor-associated cells were collected for flow cytometry and scRNA-seq analysis, and FFPE sample of tumor tissue was analyzed by a multispectral imager. The combination group demonstrated the superior anti-cancer efficacy to vehicle, pembrolizumab, and SKI-G-801 groups (all p<0.05). The scRNA-seq was performed with Seurat 4.0. The cells were separated into total 9 clusters. Following SKI-G-801 treatment, the T cells showed higher expressions of CD2, CCL5, CCL4, GZMA, and GZMB compared to both pembrolizumab and vehicle groups (both p<0.05). The gene expressions of CD2 and GZMA were higher than pembrolizumab and vehicle groups (both p<0.05). The CDR3 length differences of TCRs and diversity of T cell clones were higher increased in SKI-G-801, pembrolizumab and combination groups than vehicle group (p<0.05). In the macrophage cluster, the HLA-A, HLA-DRA, HLA-DRB1, and IL-1B genes were commonly expressed in SKI-G-801 and combination group (p<0.05). Mostly, the professional antigen-presenting macrophages (MHC class 2 protein complex high, p<0.05) were increased in the combination group. The combination effects between pembrolizumab and SKI-G-801 can be addressed by enhanced antigen presenting machinery, T cell activation and unique T cell clones. These transcriptomic results were highly correlated with the results of multispectral imaging and flow cytometry. Tumor infiltrations of helper T cells and cytotoxic T cells significantly increased in combination and SKI-G-801 groups (p < 0.01, p < 0.001 respectively). In multiplex IHC analysis, the proportion of CD4+ and CD8+ T cells were significantly increased in the tumor nest (both p<0.05), but the Tregs were not changed in the tumor nest which observations were validated by further flow cytometry analysis. A novel AXL inhibitor, SKI-G-801 drives priming of professional antigen-presenting cells and tumor-infiltrating T cells, leading to immunological synapsis for tumor killing, which is significantly enhanced by the combination with pembrolizumab. These results suggest the inhibition of AXL signal pathway by SKI-G-801 could confer a solid rationale for clinical investigation of lung cancer cells. Citation Format: Kyung-Ho Pyo, Hee Kyu Lee, Ha Ni Jo, Wongeun Lee, Seong Gu Heo, Sang Bin Lim, Dong Kwon Kim, Chun-Bong Synn, Youngseon Byeon, Young Seob Kim, Beung-Chul Ahn, Min Hee Hong, Sun Min Lim, Hye Ryun Kim, Byoung Chul Cho. Single-cell RNA sequencing reveals priming professional antigen-presenting macrophages and chemokine expressing T cells in tumor microenvironment by AXL inhibitor, SKI-G-801 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2755.

Smek1 deficiency exacerbates experimental autoimmune encephalomyelitis by activating proinflammatory microglia and suppressing the IDO1-AhR pathway

BackgroundExperimental autoimmune encephalomyelitis (EAE) is an animal disease model of multiple sclerosis (MS) that involves the immune system and central nervous system (CNS). However, it is unclear how genetic predispositions promote neuroinflammation in MS and EAE. Here, we investigated how partial loss-of-function of suppressor of MEK1 (SMEK1), a regulatory subunit of protein phosphatase 4, facilitates the onset of MS and EAE.MethodsC57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) to establish the EAE model. Clinical signs were recorded and pathogenesis was investigated after immunization. CNS tissues were analyzed by immunostaining, quantitative polymerase chain reaction (qPCR), western blot analysis, and enzyme-linked immunosorbent assay (ELISA). Single-cell analysis was carried out in the cortices and hippocampus. Splenic and lymph node cells were evaluated with flow cytometry, qPCR, and western blot analysis.ResultsHere, we showed that partial Smek1 deficiency caused more severe symptoms in the EAE model than in controls by activating myeloid cells and that Smek1 was required for maintaining immunosuppressive function by modulating the indoleamine 2,3-dioxygenase (IDO1)-aryl hydrocarbon receptor (AhR) pathway. Single-cell sequencing and an in vitro study showed that Smek1-deficient microglia and macrophages were preactivated at steady state. After MOG35-55 immunization, microglia and macrophages underwent hyperactivation and produced increased IL-1β in Smek1-/+ mice at the peak stage. Moreover, dysfunction of the IDO1-AhR pathway resulted from the reduction of interferon γ (IFN-γ), enhanced antigen presentation ability, and inhibition of anti-inflammatory processes in Smek1-/+ EAE mice.ConclusionsThe present study suggests a protective role of Smek1 in autoimmune demyelination pathogenesis via immune suppression and inflammation regulation in both the immune system and the central nervous system. Our findings provide an instructive basis for the roles of Smek1 in EAE and broaden the understanding of the genetic factors involved in the pathogenesis of autoimmune demyelination.

Gut germinal center regeneration and enhanced antiviral immunity by mesenchymal stem/stromal cells in SIV infection.

Although antiretroviral therapy suppresses HIV replication, it does not eliminate viral reservoirs or restore damaged lymphoid tissue, posing obstacles to HIV eradication. Using the SIV model of AIDS, we investigated the effect of mesenchymal stem/stromal cell (MSC) infusions on gut mucosal recovery, antiviral immunity, and viral suppression and determined associated molecular/metabolic signatures. MSC administration to SIV-infected macaques resulted in viral reduction and heightened virus-specific responses. Marked clearance of SIV-positive cells from gut mucosal effector sites was correlated with robust regeneration of germinal centers, restoration of follicular B cells and T follicular helper (Tfh) cells, and enhanced antigen presentation by viral trapping within the follicular DC network. Gut transcriptomic analyses showed increased antiviral response mediated by pathways of type I/II IFN signaling, viral restriction factors, innate immunity, and B cell proliferation and provided the molecular signature underlying enhanced host immunity. Metabolic analysis revealed strong correlations between B and Tfh cell activation, anti-SIV antibodies, and IL-7 expression with enriched retinol metabolism, which facilitates gut homing of antigen-activated lymphocytes. We identified potentially new MSC functions in modulating antiviral immunity for enhanced viral clearance predominantly through type I/II IFN signaling and B cell signature, providing a road map for multipronged HIV eradication strategies.

PH-responsive core-shell nanogels induce in situ antigen production for cancer treatment

Chemo-immunotherapy has the superiority of remedying malignant and metastatic tumors, and acquiring better therapeutic effect as well as prognosis than monotherapy. Herein, we report a kind of pH-responsive core-shell nanoparticles to achieve the co-delivery of oxaliplatin intermediate (oxa im) (HCLO NPs) and cytosine-guanine containing oligodeoxynucleotides (CpG) for cancer treatment. The nanoparticles comprise the core of lipid-coated calcium carbonate and the outer layer of hyaluronic acid (HA), providing itself with pH-sensitivity and great biocompatibility. When intratumorally injected, oxa im induces in situ antigen production through immunogenic cell death (ICD) while CpG enhances antigen presentation and facilitates the generation of cytotoxic T lymphocytes (CTLs). In vitro, the HCLO NPs enhanced the toxicity of oxa im to CT26 cells and up-regulated the expression of calreticulin (CRT, “eat me” signal). What’s more, oxaliplatin precursor (oxa pre) directly upregulated CD47 (“don’t eat me” signal) expression in CT26 cells, while being encapsulated into NPs attenuated this upregulation. In vivo, HCLO NPs elicited prominent anti-tumor immunity and abscopal effect, meanwhile significantly increased the infiltration of CD8+ T cells in the tumor microenvironment.

Synergistic enhancement of immunological responses triggered by hyperthermia sensitive Pt NPs via NIR laser to inhibit cancer relapse and metastasis

The combination of tumor ablation and immunotherapy is a promising strategy against tumor relapse and metastasis. Photothermal therapy (PTT) triggers the release of tumor-specific antigens and damage associated molecular patterns (DAMPs) in-situ. However, the immunosuppressive tumor microenvironment restrains the activity of the effector immune cells. Therefore, systematic immunomodulation is critical to stimulate the tumor microenvironment and augment the anti-tumor therapeutic effect. To this end, polyethylene glycol (PEG)-stabilized platinum (Pt) nanoparticles (Pt NPs) conjugated with a PD-L1 inhibitor (BMS-1) through a thermo-sensitive linkage were constructed. Upon near-infrared (NIR) exposure, BMS-1 was released and maleimide (Mal) was exposed on the surface of Pt NPs, which captured the antigens released from the ablated tumor cells, resulting in the enhanced antigen internalization and presentation. In addition, the Pt NPs acted as immune adjuvants by stimulating dendritic cells (DCs) maturation. Furthermore, BMS-1 relieved T cell exhaustion and induced the infiltration of effector T cells into the tumor tissues. Thus, Pt NPs can ablate tumors through PTT, and augment the anti-tumor immune response through enhanced antigen presentation and T cells infiltration, thereby preventing tumor relapse and metastasis.