Potent CD8 Research Articles

Depletion of conventional CD4+ T cells is required for robust priming and dissemination of tumor antigen-specific CD8+ T cells in the setting of anti-CD4 therapy

BackgroundOvercoming immune suppression is a major barrier to eliciting potent CD8+ T cell responses against cancer. Treatment with anti-CD4 monoclonal antibody is an effective means for eliminating CD4+Foxp3+ regulatory (Treg)...

Increases in 4-Acetaminobutyric Acid Generated by Phosphomevalonate Kinase Suppress CD8+ T Cell Activation and Allow Tumor Immune Escape.

Certain metabolites in the tumor microenvironment (TME) can alter innate immunity. Here, it is shown how phosphomevalonate kinase (PMVK) allows hepatocellular carcinoma (HCC) cells to overcome the anti-tumor immunity mediated by CD8+ T cells. In HCCs, depletion of PMVK is required to facilitate CD8+ T cell activation and their subsequent suppression of tumor growth. Mechanistically, PMVK phosphorylates and stabilizes glutamate decarboxylase 1 (GAD1), thus increasing the synthesis of γ-aminobutyric acid (GABA), which normally functions as a neurotransmitter. However, PMVK also recruits acetyl-CoA acetyltransferase 1 (ACAT1) and allows it to convert GABA, to 4-acetaminobutyric acid (4-Ac-GABA), which is released into the TME. There, 4-Ac-GABA activates the GABAA receptor (GABAAR) on CD8+ T cells, which inhibits AKT1 signaling. This in turn suppresses CD8+ T cell activation, intratumoral infiltration, and the anti-tumor response. Inhibiting PMVK or GABAAR in HCC mouse models overcomes resistance to anti-PD-1 immune checkpoint therapy. These findings reveal non-canonical and cooperative functions among the key metabolic enzymes PMVK, GAD1, and ACAT1 that reprogram glutamine metabolism to synthesize a potent CD8+ T cell inhibitor 4-Ac-GABA. Blocking 4-Ac-GABA signaling in CD8+ T cells, particularly when combined with immune checkpoint inhibition, potentially represents a new and potent form of immunotherapy.

A therapeutic HBV vaccine containing a checkpoint modifier enhances CD8+ T cell and antiviral responses.

In patients who progress from acute hepatitis B virus (HBV) infection to a chronic HBV (CHB) infection, CD8+ T cells fail to eliminate the virus and become impaired. A functional cure of CHB likely requires new and highly functional CD8+ T cell responses different from those induced by the infection. Here we report preclinical immunogenicity and efficacy of an HBV therapeutic vaccine that includes herpes simplex virus (HSV) glycoprotein D (gD), a checkpoint modifier of early T cell activation, that enhances, broadens, and prolongs CD8+ T cell responses. We developed a therapeutic HBV vaccine based on a chimpanzee adenovirus serotype 6 (AdC6) vector, called AdC6-gDHBV2, that targets conserved and highly immunogenic regions of the viral polymerase (pol) and core antigens fused into HSV gD. The vaccine was tested with, and without gD, in mice for immunogenicity and in an adeno-associated virus (AAV)8-1.3HBV vector model for antiviral efficacy. The vaccine encoding the HBV antigens within gD stimulates potent and broad CD8+ T cell responses. In a surrogate model of HBV infection, a single intramuscular (i.m.) injection of AdC6-gDHBV2 achieved significant and sustained declines of circulating HBV DNA copies (cps) and HBV surface antigen (HBsAg); both inversely correlated with HBV specific CD8+ T cell frequencies in spleens and livers. AdC6-gDHBV2 is the first therapeutic vaccine to show significant reductions in levels of HBV genome copies and HBsAg when used alone, even when vaccination was delayed for months from infection.

Therapeutic vaccine targeting dual immune checkpoints induces potent multifunctional CD8+ T cell anti-tumor immunity

BackgroundVaccines targeting immune checkpoints represent a promising immunotherapeutic approach for solid tumors. However, the therapeutic efficacy of dual targeting immune checkpoints is still unclear in renal carcinoma. MethodsAn adenovirus (Ad) vaccine targeting B7H1 and B7H3 was developed and evaluated for its therapeutic efficacy in subcutaneous, lung metastasis or orthotopic renal carcinoma mouse and humanized models using flow cytometry, Enzyme-linked immunosorbent spot (ELISPOT), cytotoxic T lymphocyte (CTL) killing, cell deletion, hematoxylin and eosin (HE) staining, and immunohistochemistry (IHC) assays. ResultsThe Ad-B7H1/B7H3 immunization effectively inhibited tumor growth and increased the induction and percentages of CD8+ T cells in subcutaneous tumor models. The vaccine enhanced the induction and maturation of CD11c+ or CD8+CD11c+ cells, promoting tumor-specific CD8+ T cell immune responses. This was evidenced by increased proliferation of CD8+ T cells and enhanced CTL killing activity. Deletion of CD8+ T cells in vivo abolished the anti-tumor effect of the Ad-B7H1/B7H3 vaccine, highlighting the pivotal role of functional CD8+ T cell immune responses. Moreover, significant therapeutic efficacy of the Ad-B7H1/B7H3 vaccine was observed in lung metastasis, orthotopic, and humanized tumor models through multifunctional CD8+ T cell immune responses. ConclusionsThe Ad vaccine targeting dual immune checkpoints B7H1 and B7H3 exerts a potent therapeutic effect for renal carcinoma and holds promise for solid tumor treatment.

The asymmetric opening of HIV-1 Env by a potent CD4 mimetic enables anti-coreceptor binding site antibodies to mediate ADCC.

HIV-1 envelope glycoproteins (Env) from primary HIV-1 isolates typically adopt a pretriggered "closed" conformation that resists to CD4-induced (CD4i) non-neutralizing antibodies (nnAbs) mediating antibody-dependent cellular cytotoxicity (ADCC). CD4-mimetic compounds (CD4mcs) "open-up" Env allowing binding of CD4i nnAbs, thereby sensitizing HIV-1-infected cells to ADCC. Two families of CD4i nnAbs, the anti-cluster A and anti-coreceptor binding site (CoRBS) Abs, are required to mediate ADCC in combination with the indane CD4mc BNM-III-170. Recently, new indoline CD4mcs with improved potency and breadth have been described. Here, we show that the lead indoline CD4mc, CJF-III-288, sensitizes HIV-1-infected cells to ADCC mediated by anti-CoRBS Abs alone, contributing to improved ADCC activity. Structural and conformational analyses reveal that CJF-III-288, in combination with anti-CoRBS Abs, potently stabilizes an asymmetric "open" State-3 Env conformation, This Env conformation orients the anti-CoRBS Ab to improve ADCC activity and therapeutic potential.

Pickering emulsion-guided monomeric delivery of monophosphoryl lipid A for enhanced vaccination

Immunological adjuvants are vaccine components that enhance long-lasting adaptive immune responses to weakly immunogenic antigens. Monophosphoryl lipid A (MPLA) is a potent and safe vaccine adjuvant that initiates an early innate immune response by binding to the Toll-like receptor 4 (TLR4). Importantly, the binding and recognition process is highly dependent on the monomeric state of MPLA. However, current vaccine delivery systems often prioritize improving the loading efficiency of MPLA, while neglecting the need to maintain its monomeric form for optimal immune activation. Here, we introduce a Pickering emulsion-guided MPLA monomeric delivery system (PMMS), which embed MPLA into the oil-water interface to achieve the monomeric loading of MPLA. During interactions with antigen-presenting cells, PMMS functions as a chaperone for MPLA, facilitating efficient recognition by TLR4 regardless of the presence of lipopolysaccharide-binding proteins. At the injection site, PMMS efficiently elicited local immune responses, subsequently promoting the migration of antigen-internalized dendritic cells to the lymph nodes. Within the draining lymph nodes, PMMS enhanced antigen presentation and maturation of dendritic cells. In C57BL/6 mice models, PMMS vaccination provoked potent antigen-specific CD8+ T cell-based immune responses. Additionally, PMMS demonstrated strong anti-tumor effects against E.G7-OVA lymphoma. These data indicate that PMMS provides a straightforward and efficient strategy for delivering monomeric MPLA to achieve robust cellular immune responses and effective cancer immunotherapy.

Radioactive Hydroxyapatite Microspheres Empower Sustainable In Situ Tumor Vaccination.

Tumor in situ vaccination (ISV) strategies have emerged in clinical trials as promising approaches, involving the release of tumor antigens through local radiotherapy and intratumorally adjuvant injections. However, the current fabrication strategy for achieving a sustainable immune response to ISV remains a pressing challenge. In this study, we present an empowered sustainable ISV method for antitumor therapy using 177Lu-labeled manganese-doped mesoporous hydroxyapatite (177Lu/Mn-HAP) microspheres. The ISV enables the sustained utilization of tumor antigens, leading to the activation of dendritic cells and polarization of macrophages toward the M1 subtype. Consequently, it facilitates the generation of potent CD8+ T-cell responses, enhancing the antitumor effects of internal radiation in both primary and distant tumors. Importantly, this approach achieves complete remission in all tumor-bearing mice and stimulates immune memory to prevent tumor recurrence. Our study highlights a universal and safe ISV strategy capable of inducing potent tumor-specific and sustainable immune response.

An injectable composite hydrogel containing polydopamine-coated curcumin nanoparticles and indoximod for the enhanced combinational chemo-photothermal-immunotherapy of breast tumors

The complexity and compensatory evolution of tumors weaken the effectiveness of single antitumor therapies. Therefore, multimodal combination therapies hold great promise in defeating tumors. Herein, we constructed a multi-level regulatory co-delivery system based on chemotherapy, phototherapy, and immunotherapy. Briefly, curcumin (Cur) was prepared as nanoparticles and coated with polydopamine (PDA) to form PCur-NPs, which along with an immune checkpoint inhibitor (indoximod, IND) were then loaded into a thermosensitive Pluronic F127 (F127) hydrogel to form a multifunctional nanocomposite hydrogel (PCur/IND@Gel). The in situ-formed hydrogel exhibited excellent photothermal conversion efficiency and sustained drug release behavior both in vitro and in vivo. In addition, PCur-NPs showed enhanced cellular uptake and cytotoxicity under NIR laser irradiation and induced potent immunogenic cell death (ICD). After intratumoral injection of PCur/IND@Gel, significant apoptosis in 4T1 tumors was induced, dendritic cells in lymph nodes were highly activated, potent CD8+ and CD4+ antitumor immune responses were elicited and regulative T cells in tumors were significantly reduced, which notably inhibited the tumor growth and prolonged the survive time of 4T1 tumor-bearing mice. Therefore, this injectable nanocomposite hydrogel is a promising drug co-delivery platform for chemo-photothermal-immunotherapy of breast tumors.

Protective Efficacy of the Epitope-Conjugated Antigen N-Tc52/TSkb20 in Mitigating Trypanosoma cruzi Infection through CD8+ T-Cells and IFNγ Responses.

Chagas disease, caused by the protozoan Trypanosoma cruzi, remains a major public health challenge affecting millions in Latin America and worldwide. Although significant progress has been made in vector control, no vaccine exists to prevent infection or mitigate disease pathogenesis. We developed a rationally designed chimeric protein vaccine, N-Tc52/TSkb20, incorporating immunodominant epitopes from two T. cruzi antigens, the amino-terminal portion of Tc52 and the TSkb20 epitope derived from trans-sialidase. The objectives of this study were to construct and characterize the antigen and evaluate its protective potential in an immunoprophylactic murine model of T. cruzi infection. The N-Tc52/TSkb20 protein was recombinantly expressed in E. coli and its identity was confirmed using mass spectrometry and Western blotting. Immunization with the chimeric protein significantly controlled parasitemia and reduced the heart, colon, and skeletal muscle parasite burdens compared to non-vaccinated mice. Protection was superior to vaccination with the individual parental antigen components. Mechanistically, the vaccine induced potent CD8+ T-cell and IFNγ responses against the incorporated epitopes and a protective IgG antibody profile. A relatively low IL-10 response favored early parasite control. These results validate the promising multi-epitope approach and support the continued development of this type of rational vaccine design strategy against Chagas disease.

A Virus-Inspired Inhalable Liponanogel Induces Potent Antitumor Immunity and Regression in Metastatic Lung Tumors.

Pulmonary delivery of immunostimulatory agents such as poly(I:C) to activate double-stranded RNA sensors MDA5 and RIG-I within lung-resident antigen-presenting cells is a potential strategy to enhance antitumor immunity by promoting type I interferon secretion. Nevertheless, following pulmonary delivery, poly(I:C) suffers from rapid degradation and poor endosomal escape, thus limiting its potency. Inspired by the structure of a virus that utilizes internal viral proteins to tune the loading and cytosolic delivery of viral nucleic acids, we developed a liponanogel (LNG)-based platform to overcome the delivery challenges of poly(I:C). The LNG comprised an anionic polymer hyaluronic acid-based nanogel core coated by a lipid shell, which served as a protective layer to stabilize the nanogel core in the lungs. The nanogel core was protonated within acidic endosomes to enhance the endosomal membrane permeability and cytosolic delivery of poly(I:C). After pulmonary delivery, LNG-poly(I:C) induced 13.7-fold more IFNβ than poly(I:C) alone and two-fold more than poly(I:C) loaded in the state-of-art lipid nanoparticles [LNP-poly(I:C)]. Additionally, LNG-poly(I:C) induced more potent CD8+ T-cell immunity and stronger therapeutic effects than LNP-poly(I:C). The combination of LNG-poly(I:C) and PD-L1 targeting led to regression of established lung metastases. Due to the ease of manufacturing and the high biocompatibility of LNG, pulmonary delivery of LNG may be broadly applicable to the treatment of different lung tumors and may spur the development of innovative strategies for cancer immunotherapy. Significance: Pulmonary delivery of poly(I:C) with a virus-inspired inhalable liponanogel strongly activates cytosolic MDA5 and RIG-I and stimulates antitumor immunity, representing a promising strategy for safe and effective treatment of metastatic lung tumors.

Abstract LB076: Targeting an aging-related molecule overcomes resistance to anti-PD1 treatment of cancer

Abstract Purpose: Despite receiving great attention as a promising treatment, therapeutic efficacies induced by immune checkpoint inhibitors are low in clinical settings, and thus there are high expectations for the development of innovative diagnostic and therapeutic methods. We previously demonstrated that blocking FSTL1 overcomes anti-PD1 resistance in aged mice. In this study, we comprehensively analyzed and compared gene expressions in splenic T cells harvested from young and old mice receiving anti-FSTL1 or anti-PD1 therapy after tumor implantation using the GeneChip microarray in order to identify a key gene in the anti-PD1 resistance. In addition, we evaluated anti-tumor efficacy induced by blocking the identified molecule using aged tumor models and tumor metastasis models (3LL or Colon26), which are resistant to anti-PD1 therapy. Furthermore, to validate the clinical relevancy, we analyzed immune cells isolated from malignant tumor ascites and peripheral blood cells of gastric cancer patients by flow cytometry. Results: Using the GeneChip microarray data of splenic T cells, we identified α-synuclein (SNCA), which is a neuropathological molecule involved in Parkinson's disease and other synucleinopathies, as a gene that was significantly reduced by anti-FSTL1 therapy, but not anti-PD1 therapy, in splenic T cells of aged tumor models. SNCA+ cell subsets significantly increased not only in CD3+ T cells, but also CD11b+ myeloid cells and CD45- mesenchymal stromal/stem cells, within spleen, peripheral blood, and tumor tissues of aged mice and mouse tumor models, but rarely in those of young and naive mice. Increase of the subsets were also observed in malignant tumor ascites and peripheral blood of gastric cancer patients, but not those of healthy donors. Blocking SNCA with the specific mAb significantly induced potent CD8+ T cells with greater cytotoxicity in the in vitro CTL induction setting, and also significantly induced robust anti-tumor immunity in the in vivo therapeutic setting with anti-PD1-resistant tumor models. Combination of anti-SNCA therapy successfully elicited anti-PD1 therapeutic efficacy, providing significantly better prognosis in many tumor models, including severe tumor ascites models. Conclusions: These results suggest that SNCA is a type of immune inhibitory checkpoint molecules and an immunological determinant of anti-PD1 resistance, although the further study is needed to clarify the molecular functions in immune cells. Targeting this molecule may represent a promising strategy to overcome the anti-PD1 resistance and contribute to improved clinical outcomes in cancer treatment. Citation Format: Chie Kudo-Saito, Hiroshi Imazeki, Hirokazu Shoji, Narikazu Boku. Targeting an aging-related molecule overcomes resistance to anti-PD1 treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB076.

Preclinical evaluation of two phylogenetically distant arenavirus vectors for the development of novel immunotherapeutic combination strategies for cancer treatment

BackgroundEngineered arenavirus vectors have recently been developed to leverage the body’s immune system in the fight against chronic viral infections and cancer. Vectors based on Pichinde virus (artPICV) and lymphocytic...

Combining CRISPR-Cas9 and TCR exchange to generate a safe and efficient cord blood-derived T cell product for pediatric relapsed AML

BackgroundHematopoietic cell transplantation (HCT) is an effective treatment for pediatric patients with high-risk, refractory, or relapsed acute myeloid leukemia (AML). However, a large proportion of transplanted patients eventually die due...

Abstract 162: Fatty acid synthesis blockade synergizes with immune checkpoint blockade by repolarizing macrophages and enhancing anti-tumor cytotoxicity in gastric cancer

Abstract Background: Immune checkpoint blockade (ICB), which unleashes potent CD8+ T cell-based anti-tumor responses, has emerged as a promising therapeutic option for several cancer types. However, response rates stay limited in gastric cancer (GC) patients, indicating the necessity of combinational strategies to overcome other immunosuppressive factors. Fatty acid synthesis (FAS) is an indispensable metabolic pathway proven to support cancer cell survival. The rate-limiting enzyme of FAS, FASN, is upregulated in several cancers and could contribute to cancer immune escape. Though some investigations propose that specific gene alterations can cause FASN upregulation under the GC setting, the immunoregulatory role and therapeutic value of FASN in the tumor microenvironment (TME) of GC have not been fully elucidated. Methods: FASN expression was measured by Western Blotting and flow cytometry. Bioinformatic analysis was conducted via an online website (www.camoip.net). The syngeneic GC mouse model was established by subcutaneous inoculation of MFC cells in 615-line mice, treated by vehicles, FASN inhibitor (FASNi), anti-PD-1 antibodies, or FASNi+anti-PD-1 antibodies. TAM and CD8+ T cell depletion was implemented by anti-CSF1R and anti-CD8α antibodies. Bone marrow-derived macrophages (BMDMs) were extracted from the femurs of 615-line mice, induced by M-CSF and stimulated by LPS or IL-4. The proportions and functional status of immune cells were identified by flow cytometry. Results: FASN was upregulated in GC tissues and GC human cell lines. Bioinformatic analyses demonstrated that FASN expression was related to poor infiltration of cytotoxic lymphocytes, as well as the downregulation of pathways related to anti-cancer immune responses. FASNi inhibited tumor growth in a syngeneic GC mouse model, with elevated PD-1 and IFN-γ expression in CD8+ T cells and increased NK cell infiltration, suggesting the enhancement in anti-cancer cytotoxicity. Meanwhile, FASNi induced a shift from M2-like to M1-like in tumor-associated macrophages (TAM). We detected that M2-like TAM and CD8+ T cells harbored higher FASN expression than other immune cells in GC mouse tissues, and tumor-suppressing effects of FASN inhibition could be reversed by TAM and CD8+ T cell depletion, which corroborated the key role of TAM and CD8+ T cells in shaping FASN-mediated tumor regulation. FASNi could also repolarize BMDMs from M1-like to M2-like in vitro. Finally, FASNi exhibited synergistic tumor control with anti-PD-1 therapy in GC and melanoma mouse models. Conclusion: This work sheds light on the impact of FASN on TME modulation of GC. We proved that FASNi could repolarize M2-like TAM to M1 phenotypes and promote cytotoxic lymphocyte-mediated anti-tumor immunity. Our results propose FASN inhibition as a potential therapeutic strategy to sensitize ICB therapy in GC. Citation Format: Hanbing Wang, Tao Shi, Yue Wang, Yuting Luo, Yunfeng Pan, Xiaoyu Zhou, Yue Zhang, Baorui Liu, Jia Wei. Fatty acid synthesis blockade synergizes with immune checkpoint blockade by repolarizing macrophages and enhancing anti-tumor cytotoxicity in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 162.

Abstract 1520: A novel cluster of cytolytic alpha/beta TCR+CD4−CD8−T cells originating from CD8+T cells with potent innate-like T cell killing ability: implicating adoptive T cell transfer therapy for hepatocellular carcinoma

Abstract Tumor immunotherapies represent promising strategies for treating various cancers, including hepatocellular carcinoma. In this context, immune cells, including the adoptive T cell transfer treatment, become the focus of interests. Notably, alpha/beta TCR+CD4−CD8−T (DNT) cells is a subgroup of T cells that is ignored for a long time but was re-focused recently. These DNT cells are a group of cells with opposite functions in different condition, including pro-inflammatory or suppressive functions and possibly originated from over-stimulation of peripheral CD4+T cells/CD8+T cells or a special group of T cells formed in thymus. In our research results, we have found these DNT cells increased in murine hepatoma model, especially in the tumor microenvironment. In addition, these DNT cells from tumor microenvironment of murine hepatoma possessed potent tumor-killing ability. Furthermore, we analyzed the transcriptome profile of T cells in tumor microenvironment by single cell RNAseq approach (CITE-seq). We found DNT cells could be categorized into two main clusters, one is cytolytic DNT cells, resemble the potent CD8+T cells, and another is the CD4+T cell-like DNT cells. These cytolytic DNT cells are not exhausted but with high expression of cytolytic molecule and low hypoxia-inducible factor-1alpha (HIF-1a) expression. By trajectory analysis, cytolytic DNT cells come from the conventional CD8+T cells but following a unique developmental pathway that is different from the exhausted CD8+T cells. Interesting, these cytolytic DNT cells also expressed high level of Fcer1g molecule, a marker of innate CD8+T cells. In addition, these DNT cells could be generated and expanded in-vitro by IL-15 without TCR activation. In conclusion, we identified a cluster of cytolytic DNT cells, originated from CD8+T cells, exhibits innate T cells behavior and potent killing ability. In addition, these cytolytic DNT cells could be generated and expanded in-vitro for the adoptive T cell transfer treatment of hepatoma. Citation Format: Cheng-Heng Wu, Te-Wei Tseng, Chen-Yang Huang, Ping-Hsun Ou, Yi-Chen Lin, Pin-Jung Chen, Po-Ting Lin, Chan-Keng Yang, Wei Teng, Tsung-Han Wu, Yung-Chang Lin, Chun-Yen Lin. A novel cluster of cytolytic alpha/beta TCR+CD4−CD8−T cells originating from CD8+T cells with potent innate-like T cell killing ability: implicating adoptive T cell transfer therapy for hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1520.

Role of IL-27 in Epstein-Barr virus infection revealed by IL-27RA deficiency.

Epstein-Barr virus (EBV) infection can engender severe B cell lymphoproliferative diseases1,2. The primary infection is often asymptomatic or causes infectious mononucleosis (IM), a self-limiting lymphoproliferative disorder3. Selective vulnerability to EBV has been reported in association with inherited mutations impairing T cell immunity to EBV4. Here we report biallelic loss-of-function variants in IL27RA that underlie an acute and severe primary EBV infection with a nevertheless favourable outcome requiring a minimal treatment. One mutant allele (rs201107107) was enriched in the Finnish population (minor allele frequency = 0.0068) and carried a high risk of severe infectious mononucleosis when homozygous. IL27RA encodes the IL-27 receptor alpha subunit5,6. In the absence of IL-27RA, phosphorylation of STAT1 and STAT3 by IL-27 is abolished in T cells. In in vitro studies, IL-27 exerts a synergistic effect on T-cell-receptor-dependent T cell proliferation7 that is deficient in cells from the patients, leading to impaired expansion of potent anti-EBV effector cytotoxic CD8+ T cells. IL-27 is produced by EBV-infected B lymphocytes and an IL-27RA-IL-27 autocrine loop is required for the maintenance of EBV-transformed B cells. This potentially explains the eventual favourable outcome of the EBV-induced viral disease in patients with IL-27RA deficiency. Furthermore, we identified neutralizing anti-IL-27 autoantibodies in most individuals who developed sporadic infectious mononucleosis and chronic EBV infection. These results demonstrate the critical role of IL-27RA-IL-27 in immunity to EBV, but also the hijacking of this defence by EBV to promote the expansion of infectedtransformed B cells.

Antigen Self-Presented Personalized Nanovaccines Boost the Immunotherapy of Highly Invasive and Metastatic Tumors.

Dendritic cell (DC)-based vaccines have shown promise in adoptive cell therapy for enhancing the antigen-specific response of antitumor immunity. However, their clinical efficacy is limited by the less-presented tumor-associated antigens (TAAs) through MHC I and low lymph node homing efficiency. Herein, to address these issues, we rationally design and fabricate DC-based nanovaccines by coating Cu2-xSe nanoparticles (CS NPs) with the membrane of matured DCs (named as DCNV(CSD) nanovaccines). We reveal the important roles of CS NPs in the DCNV(CSD) nanovaccines from three aspects: (1) inducing the immunogenic cell death of tumor cells to expose abundant TAAs; (2) promoting the escape of TAAs from the lysosomes of DCs during the antigen presenting process through MHC I; (3) sustainably releasing traces of copper ions to promote the proliferation of T cells. Our DCNV(CSD) nanovaccines are characterized with high expressions of MHC I, CD80, CD86, CCR7, and ICAM-1 proteins, which not only endow them with abundantly processed specific TAAs, but also a strong capability of homing to the lymph nodes. The homing capability of our small DCNV(CSD) nanovaccines is better than that of matured DCs. More importantly, they can elicit the strong response of potent antispecific CD8+ T cells for antitumor immunotherapy, as tested in the treatment of highly invasive glioblastoma and highly metastatic melanoma. Additionally, DCNV(CSD) nanovaccines can generate memory T cells (TEM) in the spleen of mice to effectively prevent the recurrence of treated tumors. This work demonstrates a universal approach to fabricate high-performance DC-based nanovaccines for tumor immunotherapy by using versatile CS NPs.

Regulation of IL-10 expression and function by JAK-STAT in CD8+ T cells

IL-10 is a pleiotropic cytokine that plays a significant role in antiviral and antitumor immunity. Potent CD8+ T cells express IL-10 after stimulation by strong TCR signaling, which promotes the killing effect of CD8+ T cells. However, the regulation of IL-10 expression in CD8+ T cells and its signaling pathway to enhance CD8+ T cell function are largely unknown. In this study, we investigated the JAK-STAT signaling molecules that regulate IL-10 expression in CD8+ T cells and the JAK-STAT signaling pathway that IL-10 enhances the function of CD8+ T cells through its receptor, using small molecule inhibitors and CRISPR-Cas9 gene editing. Our findings provide new insights and a theoretical basis for the immunotherapy of tumors.

Immune Modulation of Innate and Adaptive Responses Restores Immune Surveillance and Establishes Antitumor Immunologic Memory.

Current immunotherapies have proven effective in strengthening antitumor immune responses, but constant opposing signals from tumor cells and the surrounding microenvironment eventually lead to immune escape. We hypothesized that in situ release of antigens and regulation of both the innate and adaptive arms of the immune system would provide a robust and long-term antitumor effect by creating immunologic memory against tumors. To achieve this, we developed CARG-2020, a genetically modified virus-like vesicle (VLV) that is a self-amplifying RNA with oncolytic capacity and encodes immune regulatory genes. CARG-2020 carries three immune modulators: (i) the pleiotropic antitumor cytokine IL12, in which the subunits (p35 and p40) are tethered together; (ii) the extracellular domain (ECD) of the protumor IL17RA, which serves as a dominant-negative antagonist; and (iii) a shRNA targeting PD-L1. Using a mouse model of ovarian cancer, we demonstrated the oncolytic effect and immune-modulatory capacities of CARG-2020. By enhancing IL12 and blocking IL17 and PD-L1, CARG-2020 successfully reactivated immune surveillance by promoting M1, instead of M2, macrophage differentiation, inhibiting MDSC expansion and establishing a potent CD8+ T cell-mediated antitumoral response. Furthermore, we demonstrated that this therapeutic approach provided tumor-specific and long-term protection against the establishment of new tumors. Our results provide a rationale for the further development of this platform as a therapeutic modality for ovarian cancer patients to enhance antitumor responses and prevent a recurrence.

Potent CD8+ T-Cell Proliferation and Effector Differentiation Following Subcutaneous (SC) Mosunetuzumab Administration in Patients with Untreated, High Tumor-Burden Follicular Lymphoma (FL)

Introduction: Bispecific antibodies (BsAb) have emerged as a potent therapeutic option for patients with B-cell non-Hodgkin lymphoma, including FL. These drugs leverage endogenous CD3+ T-cells to enable cytotoxic tumor cell killing. While their mode of action has been hypothesized in in vitro and animal models, the mechanisms by which these drugs promote both initial and long-term immune-dependent disease control remains unknown. We recently initiated a multicenter phase 2 trial of the CD20xCD3 BsAb mosunetuzumab (mosun) as first-line therapy for patients with high-burden FL (NCT05389293). As part of this study, we performed serial sampling of both blood and lymph node tissues in order to elucidate T-cell changes associated with response and outcome. Here we present the first analysis of peripheral blood immune profiling. Methods: Patients with untreated, high-burden stage 2-4 FL in need of treatment received SC mosun at the dose of 5 mg on D1 and 45 mg on D8 and D15 of C1, and 45 mg on D1 of each subsequent 21-day cycle. Peripheral blood samples were obtained on C1D1, C1D8, C1D15, C2D1, C3D1, C5D1, and end of treatment (EOT, 24w from C1D1). Peripheral blood mononuclear cells (PBMCs) and plasma were isolated and cryopreserved. Samples were later thawed in batches and processed as follows: CITE-Seq + single-cell TCR sequencing was performed on viable CD11b-/CD19-/CD56-/CD3+ T-cells using the 10x Genomics Single Cell Immune Profiling Platform. RNA was extracted from 1 x 10 6 viable PBMCs using TriZol, followed by phenol/chloroform-based extraction, followed by bulk TCR sequencing using an in-house platform developed by MSKCC's Integrated Genomics Operation. The remaining cells were stained with antibodies targeting CD45RA, CD8, CD45, CD45RO, CD38, CD27, CD28, CXCR3, LAG-3, CD3, CD4, HLA-DR, CXCR5, CCR6, CD56, CD11b, CD19, CD39, PD-1, OX40, Tim-3, CCR4, Granzyme B, T-bet, FoxP3, Ki-67, and CTLA-4 and analyzed using a Cytek Aurora Spectral Cytometer. Flow cytometry analysis was performed using OMIQ. Statistical analyses were performed using a paired t-test. Results: A total of 45 samples obtained from 8 patients were analyzed, including samples from C1D1 (8), C1D8 (6), C1D15 (3), C2D1 (7), C3D1 (8), C5D1 (6), and EOT (7). High dimensional clustering of spectral cytometry data using the FlowSOM algorithm revealed 9 clusters of CD8+ T-cells corresponding to known CD8+ T-cell subsets. Among these subsets, we observed two distinct temporal patterns of CD8+ T-cells within the blood. On C1D8 there was a profound drop in circulating effector CD8+ T-cells as well as terminally differentiated CD8+ Temra cells (Figure 1), with a corresponding enrichment of naïve CD8+ T-cells (Figure). We also observed significant proliferation of newly activated T-cells on C1D8, followed by expansion of CD8+ T-cells expressing several activation and exhaustion markers including PD-1, Tim-3, and CD39, peaking on C2D1 (Figure 2). CD4+ T-cell responses were more modest with a small early increase in CD28+OX40+PD-1+ CD4+ T-cells on C1D8 and a drop in circulating GzmB+OX40+ CD4+ T-cells on C1D15. Conclusion: SC mosun therapy for newly diagnosed FL was associated with a unique peripheral blood immune profile, with early mobilization of pre-differentiated CD8+ T-cell effector cells and subsequent activation and expansion of newly primed CD8+ T-cells. Further immune phenotyping, including scRNA-seq and TCR clonal dynamics in the peripheral blood over time, as well as TCR clonality of distinct CD8+ and CD4+ T-cell populations are currently being analyzed on these and additional samples, and will be presented in detail.