Tumor-specific Immunity Research Articles

Combined delivery of IL12 and an IL18 mutant without IL18BP-binding activity by an adenoviral vector enhances tumor specific immunity

Cytokines play pivotal roles in anticancer immune response. We previously reported that adenovirus armed with an IL18 variant (DR18) that overcomes IL18BP neutralizing effect displayed powerful therapeutic effects in local and distant tumors when delivered intratumorally. Here, we tested a combined delivery of IL12 and DR18 in tumor models since IL12 and IL18 are known to act synergistically in potentiating IFNγ production and antitumor immunity. To minimize adverse effects associated with systemic delivery, we constructed oncolytic adenoviruses (oAd) harboring DR18 and IL12 (oAd.DR18/IL12). IL12 was expressed as a single chain IL12 (scIL12) peptide composed of the IL12/p40 and IL12/p35 subunits. Intratumoral administration of oAd.DR18/IL12, oAd-expressing DR18 (oAd.DR18), or oAd-expressing IL12 (oAd.IL12) showed antitumor effect in syngeneic colorectal tumor models. Compared to oAd.DR18 or oAd.IL12, administration of oAd.DR18/IL12 improved the antitumor effects as well as increased survival rate in these models. We detected enhanced tumor infiltrating T lymphocytes and NK cells in oAd.DR18/IL12-treated mice than those from mock-treated or individually treated groups. Moreover, mice received oAd.DR18/IL12 had more robust tumor-specific cytotoxicity. Importantly, mice that had tumor regression after oAd.DR18/IL12 treatment established anti-tumor specific immune memory. These results show that adenovirus armed with engineered cytokines boosts tumor specific immunity and antitumor effect.

High-intensity focused ultrasound ablation to increase tumor-specific lymphocytes in prostate cancer.

Treatment options for localized prostate cancer have been expanded by FDA-approval of High-Intensity Focused Ultrasound (HIFU). Prostate cancer typically has few tumor-infiltrating lymphocytes, which are crucial for antitumor immunity. This study investigated the use of HIFU to increase lymphocyte infiltration into the tumor and enhance antitumor immunity. RM1 prostate tumors were implanted onto both flanks of syngeneic C57BL/6 J mice, with one tumor subjected to HIFU treatment. The growth of the contralateral tumor was monitored. Blood samples were obtained from patients both before and after prostatectomy or HIFU treatment. Peripheral blood mononuclear cells (PBMCs) were then isolated to analyze the immune cells. In murine experiments, the application of HIFU to one tumor decreased the growth of the contralateral (non-HIFU treated) tumor, when the contralateral tumor was the same tumor type, but not when it was a different tumor type. HIFU increased infiltration of CD4+ and CD8+ lymphocytes into the contralateral, same-type tumor. Lymphocyte depletion studies affirmed that the antitumor immune response triggered by HIFU relies on CD4+ and CD8+ lymphocytes. Addition of cholesterol-lowering intervention further increased antitumor immunity generated by HIFU in mice. In human subjects, HIFU, but not prostatectomy, stimulated anti-tumor CD4+ and CD8+ lymphocytes. We concluded that HIFU induced a potent cellular antitumor immune response that inhibited the progression of murine prostate tumors. HIFU stimulated tumor-specific cellular immunity in patients. Future clinical trials should explore the clinical benefits of HIFU, possibly in combination with existing immunotherapies, as immune modulators for both localized and metastatic disease.

Cancer vaccines compensate for the insufficient induction of protective tumor-specific immunity of CD3 bispecific antibody therapy

BackgroundCD3 bispecific antibody (CD3 bsAb) therapy has become an established treatment modality for some cancer types and exploits endogenous T cells irrespective of their specificity. However, durable clinical responses are...

Efficient Biomarker for Immunotherapy: Measuring Broad Clones Effector Tumor Antigen-Specific T Cells in the Blood of Esophageal Cancer Patients.

Cancer is the result of the interactions between tumor cells and tumor-specific immune responses. The current biomarkers detect tumor cells' properties, but accurate measurement of tumor-specific immunity is lacking. Most immunotherapies work by activating new effector tumor antigen-specific T cells (ETASTs) or reactivating pre-existing ETASTs' repertoire. The responses to immunotherapy depend on the increase of ETASTs. The amount of ETASTs, especially in blood, is critical for therapeutic efficacy. Distinguishing ETASTs from other T cells by their structural characteristics is difficult. Therefore, nanoparticles loading whole tumor antigens are utilized to activate broad clones ETASTs pre-existing in peripheral blood, followed by detecting them. Thus, the differences between ETASTs and other T cells are transformed to the differences between activated states and unactivated states. By measuring the markers of activated states and cytotoxic functions, we can distinguish ETASTs from other T cells. Nanoparticles loading mixed multiple allogeneic tumor tissue lysates or mixed multiple tumor cell lines can be utilized as universal nanoparticles to replace nanoparticles loading personalized tumor tissue. ETASTs (TATAN-activated CD8+IFN-γ+) in esophageal cancer patients are more than those in healthy people. Measurement of the ETASTs in the blood of esophageal cancer patients before and after ongoing therapy showed that ETATSs increased in the blood of patients who were responsive to immunotherapy but did not increase in the blood of nonresponders. These illustrated that therapeutic efficacy was positively correlated with the level of ETASTs in PBMC. Altogether, this study provides us a highly accurate and specific biomarker for predicting the therapeutic efficacy of cancer immunotherapy and potentially other therapies, such as radiotherapy.

Efficient Predictor for Immunotherapy Efficacy: Detecting Pan-Clones Effector Tumor Antigen-Specific T Cells in Blood by Nanoparticles Loading Whole Tumor Antigens.

Cancer involves tumor cells and tumor-specific immunity. The ability to accurately quantify tumor-specific immunity is limited. Most immunotherapies function by activating new effector tumor antigen-specific T cells (ETASTs) or reactivating the pre-existing ETASTs repertoire. Therefore, the amount of ETASTs can be used to characterize immunotherapy efficacy. Tumor antigens are highly heterogeneous and detecting most ETASTs is challenging. Therefore, nanoparticles loading whole-cell tumor antigens are used to activate and detect pan-clones ETASTs in the blood. The differences between ETASTs and other T cells are transformed into activated and non-activated states. By measuring markers of the activated status and cytotoxic function of ETASTs, it can distinguish ETASTs from other T cells. ETASTs in patients with lung cancer are higher than those in healthy individuals and those with benign pulmonary nodules. Therapeutic efficacy positively correlated with the number of ETASTs in the blood. ETATS levels increase only in the blood of patients who respond to immunotherapy. Single-cell sequencing studies validated these findings. This study provides a highly accurate, specific, non-invasive, and efficient biomarker for predicting immunotherapy efficacy in lung and other cancers. This method can also be applied to evaluate the efficacy of other treatments, such as radiotherapy, oncolytic viruses, and nanomedicine-based therapies.

Hyper-Interferon Sensitive Influenza Induces Adaptive Immune Responses and Overcomes Resistance to Anti-PD-1 in Murine Non-Small Cell Lung Cancer.

Despite recent advances in immunotherapy with immune checkpoint inhibitors, many patients with non-small cell lung cancer (NSCLC) fail to respond or develop resistance after an initial response. In situ vaccination (ISV) with engineered viruses has emerged as a promising antigen-agnostic strategy that can both condition the tumor microenvironment and augment antitumor T-cell responses to overcome immune resistance. We engineered a live attenuated viral vaccine, hyper-IFN-sensitive (HIS) virus, by conducting a genome-wide functional screening and introducing eight IFN-sensitive mutations in the influenza genome to enhance host IFN response. Compared with wild-type influenza, HIS replication was attenuated in immunocompetent hosts, enhancing its potential as a safe option for cancer therapy. HIS ISV elicited robust yet transient type I IFN responses in murine NSCLCs, leading to an enrichment of polyfunctional effector Th1 CD4+ T cells and cytotoxic CD8+ T cells into the tumor. HIS ISV demonstrated enhanced antitumor efficacy compared with wild-type in multiple syngeneic murine models of NSCLC with distinct driver mutations and varying mutational burden. This efficacy was dependent on host type 1 IFN responses and T lymphocytes. HIS ISV overcame resistance to anti-PD-1 in LKB1-deficient murine NSCLC, resulting in improved overall survival and systemic tumor-specific immunity. These studies provide compelling evidence to support further clinical evaluation of HIS as an "off-the-shelf" ISV strategy for patients with NSCLC refractory to immune checkpoint inhibitors.

Synergy Effects of HPV E6-E7 Encoding mRNA and Nucleic Acid Immunostimulators Improve Therapeutic Potential in TC-1 Graft Tumor.

Cervical cancer is the second most common cancer among women globally and the most prevalent cancer in developing countries, which was caused by human papillomavirus (HPV) infection. Messenger RNA (mRNA) vaccines have opened up new avenues for vaccine development and pandemic preparedness with potent scalability, which may possess the potential antitumor effects of an mRNA-HPV therapeutic vaccine containing nononcogenic E6 and E7 proteins. Here, we reported a lipid nanoparticle (LNP) plus nucleic acid immunostimulators (CPG 1018 and Poly I:C) mRNA vaccine platform. The LNP-CPG 1018 capsulated HPV E6-E7 mRNA significantly promoted the maturation of bone marrow-derived dendritic cells (BMDC) in vitro and were capable of efficiently migrating to lymph nodes (LN) in vivo. In TC-1 tumor-bearing mice, the subcutaneous immunization of LNP-CPG 1018 capsulated HPV E6-E7 mRNA elicited robust tumor-specific T-cell immunity, reshaped the tumor microenvironment, and inhibited tumor growth. In conclusion, the LNP-CPG 1018 system is a promising delivery platform for facilitating the development of HPV E6-E7 mRNA cancer vaccines.

Association of glutaminase expression with immune-suppressive tumor microenvironment, clinicopathologic features, and clinical outcomes in endometrial cancer

ObjectiveIncreased glutamine metabolism by cancer cells via upregulation of the drug-targetable enzyme glutaminase may contribute to an immune-suppressive tumor microenvironment. Inhibiting glutamine metabolism can not only suppress tumor growth, but...

Novel bispecific antibody-drug conjugate targeting PD-L1 and B7-H3 enhances antitumor efficacy and promotes immune-mediated antitumor responses

BackgroundAntibody-drug conjugates (ADCs) offer a promising approach, combining monoclonal antibodies with chemotherapeutic drugs to target cancer cells effectively while minimizing toxicity.MethodsThis study examined the therapeutic efficacy and potential mechanisms of...

Enhancing Tumor-Specific immunity with SLdacA: A attenuated Salmonella-mediated c-di-AMP delivery system targeting the STING pathway

The STING agonist stimulates an anti-tumor immune response by activating T cells, but its limited tumor-targeting specificity poses risks of cytokine storms or autoimmune reactions. Conversely, attenuated Salmonella typhimurium △ppGpp (S.t△ppGpp) exhibits superior tumor-targeting specificity and potent anti-tumor immunogenicity. However, the anti-tumor effects of Salmonella carrying STING agonists remain underexplored. In this study, we engineered a strain called SLdacA, utilizing S.t△ppGpp as a carrier, to produce c-di-AMP. This engineered strain effectively enhances dendritic cell maturation and M1-type macrophage polarization by inducing type I interferon production, thereby recruiting and activating effector T cells against tumor progression. This process is regulated by the STING/type I interferon pathway. Our findings indicate that utilizing S.t△ppGpp as a delivery vehicle for STING agonists holds promise as a strategy for synergistic bacterial-mediated immunotherapy.

Carbohydrate-Lectin Interactions Reprogram Dendritic Cells to Promote Type 1 Anti-Tumor Immunity.

Cancer vaccine development is inhibited by a lack of strategies for directing dendritic cell (DC) induction of effective tumor-specific cellular immunity. Pathogen engagement of DC lectins and toll-like receptors (TLRs) is thought to shape immunity by directing T cell function. Controlling downstream responses, however, remains a major challenge. A critical goal in advancing vaccine development involves the identification of receptors that drive type 1 cellular immunity. The immune system monitors cells for aberrant glycosylation (a sign of a foreign entity), but potent activation occurs when a second signal, such as single-stranded RNA or lipopolysaccharide, is present to activate TLR signaling. To exploit dual signaling, we engineered a glycan-costumed virus-like particle (VLP) vaccine that displays a DC-SIGN-selective aryl mannose ligand and encapsulates TLR7 agonists. These VLPs deliver programmable peptide antigens to induce robust DC activation and type 1 cellular immunity. In contrast, VLPs lacking this critical DC-SIGN ligand promoted DC-mediated humoral immunity, offering limited tumor control. Vaccination with glycan-costumed VLPs generated tumor antigen-specific Th1 CD4+ and CD8+ T cells that infiltrated solid tumors, significantly inhibiting tumor growth in a murine melanoma model. The tailored VLPs also afforded protection against the reintroduction of tumor cells. Thus, DC lectin-driven immune reprogramming, combined with the modular programmability of VLP platforms, provides a promising framework for directing cellular immunity to advance cancer immunotherapies and vaccines.

Type I interferon signaling induces melanoma cell-intrinsic PD-1 and its inhibition antagonizes immune checkpoint blockade

Programmed cell death 1 (PD-1) is a premier cancer drug target for immune checkpoint blockade (ICB). Because PD-1 receptor inhibition activates tumor-specific T-cell immunity, research has predominantly focused on T-cell-PD-1 expression and its immunobiology. In contrast, cancer cell-intrinsic PD-1 functional regulation is not well understood. Here, we demonstrate induction of PD-1 in melanoma cells via type I interferon receptor (IFNAR) signaling and reversal of ICB efficacy through IFNAR pathway inhibition. Treatment of melanoma cells with IFN-α or IFN-β triggers IFNAR-mediated Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling, increases chromatin accessibility and resultant STAT1/2 and IFN regulatory factor 9 (IRF9) binding within a PD-1 gene enhancer, and leads to PD-1 induction. IFNAR1 or JAK/STAT inhibition suppresses melanoma-PD-1 expression and disrupts ICB efficacy in preclinical models. Our results uncover type I IFN-dependent regulation of cancer cell-PD-1 and provide mechanistic insight into the potential unintended ICB-neutralizing effects of widely used IFNAR1 and JAK inhibitors.

Engineered mitochondria exert potent antitumor immunity as a cancer vaccine platform

The preferable antigen delivery profile accompanied by sufficient adjuvants favors vaccine efficiency. Mitochondria, which feature prokaryotic characteristics and contain various damage-associated molecular patterns (DAMPs), are easily taken up by phagocytes and simultaneously activate innate immunity. In the current study, we established a mitochondria engineering platform for generating antigen-enriched mitochondria as cancer vaccine. Ovalbumin (OVA) and tyrosinase-related protein 2 (TRP2) were used as model antigens to synthesize fusion proteins with mitochondria-localized signal peptides. The lentiviral infection system was then employed to produce mitochondrial vaccines containing either OVA or TRP2. Engineered OVA- and TRP2-containing mitochondria (OVA-MITO and TRP2-MITO) were extracted and evaluated as potential cancer vaccines. Impressively, the engineered mitochondria vaccine demonstrated efficient antitumor effects when used as both prophylactic and therapeutic vaccines in murine tumor models. Mechanistically, OVA-MITO and TRP2-MITO potently recruited and activated dendritic cells (DCs) and induced a tumor-specific cell-mediated immunity. Moreover, DC activation by mitochondria vaccine critically involves TLR2 pathway and its lipid agonist, namely, cardiolipin derived from the mitochondrial membrane. The results demonstrated that engineered mitochondria are natively well-orchestrated carriers full of immune stimulants for antigen delivery, which could preferably target local dendritic cells and exert strong adaptive cellular immunity. This proof-of-concept study established a universal platform for vaccine construction with engineered mitochondria bearing alterable antigens for cancers as well as other diseases.

Rhodium(III) Complex Noncanonically Potentiates Antitumor Immune Responses by Inhibiting Wnt/β-Catenin Signaling.

Metal-based chemoimmunotherapy has recently garnered significant attention for its capacity to stimulate tumor-specific immunity beyond direct cytotoxic effects. Such effects are usually caused by ICD via the activation of DAMP signals. However, metal complexes that can elicit antitumor immune responses other than ICD have not yet been described. Herein, we report that a rhodium complex (Rh-1) triggers potent antitumor immune responses by downregulating Wnt/β-catenin signaling with subsequent activation of T lymphocyte infiltration to the tumor site. The results of mechanistic experiments suggest that ROS accumulation following Rh-1 treatment is a critical trigger of a decrease in β-catenin and enhanced secretion of CCL4, a key mediator of T cell infiltration. Through these properties, Rh-1 exerts a synergistic effect in combination with PD-1 inhibitors against tumor growth in vivo. Taken together, our work describes a promising metal-based antitumor agent with a noncanonical mode of action to sensitize tumor tissues to ICB therapy.

The roles of tertiary lymphoid structures in genitourinary cancers: molecular mechanisms, therapeutic strategies, and clinical applications.

The presence of tertiary lymphoid structures (TLSs) associated with distinct treatment efficacy and clinical prognosis has been identified in various cancer types. However, the mechanistic roles and clinical implications of TLSs in genitourinary (GU) cancers remain incompletely explored. Despite their potential role as predictive markers described in numerous studies, it is essential to comprehensively evaluate the characteristics of TLSs, including drivers of formation, structural foundation, cellular compositions, maturation stages, molecular features, and specific functionality to maximize their positive impacts on tumor-specific immunity. The unique contributions of these structures to cancer progression and biology have fueled interest in these structures as mediators of antitumor immunity. Emerging data are trying to explore the effects of therapeutic interventions targeting TLSs. Therefore, a better understanding of the molecular and phenotypic heterogeneity of TLSs may facilitate the development of TLSs-targeting therapeutic strategies to obtain optimal clinical benefits for GU cancers in the setting of immunotherapy. In this review, the authors focus on the phenotypic and functional heterogeneity of TLSs in cancer progression, current therapeutic interventions targeting TLSs and the clinical implications and therapeutic potential of TLSs in GU cancers.

CD40 agonist engineered immunosomes modulated tumor microenvironment and showed pro-immunogenic response, reduced toxicity, and tumor free survival in mice bearing glioblastoma

CD40 agonist antibodies (αCD40) have shown promising anti-tumor response in both preclinical and early clinical studies. However, its systemic administration is associated with immune- and hepato-toxicities which hampers its clinical usage. In addition, αCD40 showed low tumor retention and induced PD-L1 expression which makes tumor microenvironment (TME) immunosuppressive. To overcome these issues, in this study, we have developed a multifunctional Immunosome where αCD40 is conjugated on the surface and RRX-001, a small molecule immunomodulator was encapsulated inside it. Immunosomes showed higher tumor accumulation till 96 h of administration and displayed sustained release of αCD40 in vivo. Immunosomes significantly delayed tumor growth and showed tumor free survival in mice bearing GL-261 glioblastoma by increasing the population of CD45+CD8+ T cells, CD45+CD20+ B cells, CD45+CD11c+ DCs and F4/80+CD86+ cells in TME. Immunosome significantly reduced the population of T-regulatory cells, M2 macrophage, and MDSCs and lowered the PD-L1 expression. Moreover, Immunosomes significantly enhanced the levels of Th1 cytokines (IFN-γ, IL-6, IL-2) over Th2 cytokines (IL-4 and IL-10) which supported anti-tumor response. Most interestingly, Immunosomes averted the in vivo toxicities associated with free αCD40 by lowering the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), IL-6, IL-1α and reduced the degree of liver damage. In addition, Immunosomes treated long-term surviving mice showed tumor specific immune memory response which prevented tumor growth upon rechallenge. Our results suggested that this novel formulation can be further explored in clinics to improve in vivo anti-tumor efficacy of αCD40 with long-lasting tumor specific immunity while reducing the associated toxicities.

RNA-encoded Interleukin 2 with Extended Bioavailability Amplifies RNA Vaccine-Induced Antitumor T-cell Immunity.

Interleukin 2 (IL-2) is a crucial cytokine in T-cell immunity, with a promising potential in cancer vaccines. However, therapeutic application of IL-2 is hampered by its short half-life and substantial toxicity. This study reports preclinical characterization of a mouse serum albumin-IL-2 fusion protein (Alb-IL2) encoded on nucleoside-modified RNA that is delivered via a nanoparticle formulation (Alb-IL2 RNA-NP) mediating prolonged cytokine availability. Alb-IL2 RNA-NP was combined with RNA-lipoplex (RNA-LPX) vaccines to evaluate its effect on the expansion of vaccine-induced antigen specific T-cell immunity. In mice dosed with Alb-IL2 RNA-NP, translated protein was shown to be systemically available up to 2 days, with an albumin-dependent preferred presence in the tumor and tumor-draining lymph node. Alb-IL2 RNA-NP administration prolonged serum availability of the cytokine compared with murine recombinant IL-2. In combination with RNA-LPX vaccines, Alb-IL2 RNA-NP administration highly increased the expansion of RNA-LPX vaccine-induced CD8+ T cells in the spleen and blood. The combination enhanced and sustained the fraction of IL-2 receptor (IL-2R) α-positive antigen-specific CD8+ T cells and ameliorated the functional capacity of the CD8+ T-cell population. Alb-IL2 RNA-NP strongly improved the antitumor activity and survival of concomitant RNA-LPX vaccination and PD-L1 blockade in a subcutaneous mouse tumor model. The favorable pharmacokinetic properties of Alb-IL2 RNA-NP render it an attractive modality for rationally designed combination immunotherapy. RNA vaccines that induce tumor-specific T-cell immunity for Alb-IL2 RNA-NP to further amplify are particularly attractive combination partners.

Combination Immunotherapy with Vaccine and Oncolytic HSV Virotherapy Is Time Dependent.

Oncolytic virotherapy or immunovirotherapy is a strategy that utilizes viruses to selectively infect and kill tumor cells while also stimulating an immune response against the tumor. Early clinical trials in both pediatric and adult patients using oncolytic herpes simplex viruses (oHSV) have demonstrated safety and promising efficacy; however, combinatorial strategies designed to enhance oncolysis while also promoting durable T-cell responses for sustaining disease remission are likely required. We hypothesized that combining the direct tumor cell killing and innate immune stimulation by oHSV with a vaccine that promotes T cell-mediated immunity may lead to more durable tumor regression. To this end, we investigated the preclinical efficacy and potential synergy of combining oHSV with a self-assembling nanoparticle vaccine codelivering peptide antigens and Toll-like receptor 7 and 8 agonists (referred to as SNAPvax),which induces robust tumor-specific T-cell immunity. We then assessed how timing of the treatments (i.e., vaccine before or after oHSV) impacts T-cell responses, viral replication, and preclinical efficacy. The sequence of treatments was critical, as survival was significantly enhanced when the SNAPvax vaccine was given prior to oHSV. Increased clinical efficacy was associated with reduced tumor volume and increases in virus replication and tumor antigen-specific CD8+ T cells. These findings substantiate the criticality of combination immunotherapy timing and provide preclinical support for combining SNAPvax with oHSV as a promising treatment approach for both pediatric and adult tumors.

Feasibility study of a personalized tumor membrane vesicle vaccine immunotherapy for head and neck squamous cell carcinoma (HNSCC).

e14673 Background: Head and neck cancer exhibits a high degree of heterogeneity, with lack of common therapeutic targets. Our phase 1 clinical trial will evaluate a novel personalized immunotherapy approach using tumor membrane vesicles (TMV) prepared from the patients’ own surgically excised tumor tissue and modifying them by attaching GPI-B7-1 and GPI-IL-12 as immune boosting molecules. This personalized vaccine will then be administered to boost the tumor-specific immunity. We report here the feasibility of developing an effective personalized therapeutic cancer vaccine from surgically collected tumor tissue from patients with HNSCC. Methods: After obtaining informed consent, a total of 10 tumor specimens from 10 patients with HNSCC of various sites were collected (0.29-1.23 grams) and graded for cellularity by a pathologist. Tumor samples were then homogenized using sterile, disposable probes. The homogenate was ultracentrifuged over a sucrose gradient to enrich tumor membrane vesicles (TMV). Yield was evaluated by protein concentration and particle size was assessed by dynamic light scattering. TMV preparations were incorporated with GPI-B7-1 and GPI-IL-12 to generate the TMV vaccine using protein transfer, a spontaneous process. For the in vivo murine studies, TMV vaccine is prepared from murine oral cancer (MOC1) tumors and incorporated with murine GPI-B7-1 and GPI-IL-12. To investigate the protective anti-tumor immune response of TMV vaccine in combination with anti-PD1 antibody therapy, we used established murine oral cancer models. We administered two doses of TMV vaccine to naïve C57BL/6 mice and four doses of anti-PD-1 mAb. Mice were monitored for 21 months and then challenged with MOC1 tumor cells 1 week prior to euthanasia. Spleens were flash frozen in liquid nitrogen and stored in -80oC until RNA is isolated for RNA-seq analysis. Results: The yields of TMV were 2.5 – 5 mg per gram of tumor tissue from four human tumor samples. Further refinement of the TMV production process yielded up to 6 mg of TMV/gram of tumor tissue. All samples passed for incorporation of GPI-B7-1 and GPI-IL-12. GPI-B7-1 is able to bind to a natural ligand, CTLA-4 suggesting it is biologically active. All samples also demonstrated biologic activity for GPI-IL-12 as tested using a reporter cell line for IL-12 and displayed acceptable endotoxin levels in a range of 1-34 EU/mg TMV vaccine. The remaining 6 tumor samples are being processed. RNA-seq data from mouse model suggests that the TMV vaccine showed synergistic activity when combined with anti-PD1 antibody, with enhanced T cell responses. Conclusions: TMV vaccine production is feasible for a phase 1 clinical trial for patients whose resected tumor is at least 0.5 g in weight and 40% cellularity. Data in mice show that TMV vaccine synergizes with anti-PD1 in inducing tumor responses.

Effect of regulatory B cell expansion on tissue-resident TPH cells immune evasion in hepatocellular carcinoma.

e16252 Background: Within pathologically inflamed nonlymphoid tissues, TPH cells are uniquely positioned to stimulate B cell responses and antibody production. The phenotype, function and clinical relevance of TPH cells in hepatocellular carcinoma (HCC), however, are presently unknown. Methods: Flow cytometry was used to quantify the expression, phenotype and function of TPH cells. Log-rank tests were performed to evaluate disease-free survival and overall survival in samples from 39 patients and 101 patients with HCC. TPH cells, CD19+ B cells and TFH cells were cultured separately in vitro or isolated from C57/B6L mice in vivo for functional assays. Results: TPH cells highly infiltrated in tumor tissues, which influenced tumor sizes and early recurrence predictions and decreased survival times. The tumor-infiltrated TPH cells showed unique ICOShiCXCL13+IL-21-MAF+BCL-6- factors and triggered naïve B cell differentiation into regulatory B cells. Triggering PD-1 induced the production of CXCL13 by TPH cells, which then suppressed tumor-specific immunity and promoted disease progression. Conclusions: Our study reveals a novel regulatory mechanism of TPH cell-regulatory B cell mediated immunosuppression and provides an important perspective for the differentiation balance between protumorigenic TPH cells and antitumorigenic TFH cells in the HCC microenvironment.