Cytotoxic T-lymphocyte Responses Research Articles

PD-1 knockout on cytotoxic primary murine CD8+ T cells improves their motility in retrovirus infected mice

Cytotoxic T lymphocyte (CTL) motility is an important feature of effective CTL responses and is impaired when CTLs become exhausted, e.g. during chronic retroviral infections. A prominent T cell exhaustion marker is programmed cell death protein 1 (PD-1) and antibodies against the interaction of PD-1 and PD-ligand 1 (PD-L1) are known to improve CTL functions. However, antibody blockade affects all PD-1/PD-L1-expressing cell types, thus, the observed effects cannot be attributed selectively to CTLs. To overcome this problem, we performed CRISPR/Cas9 based knockout of the PD-1 coding gene PDCD1 in naïve Friend Retrovirus (FV)-specific CTLs. We transferred 1,000 of these cells into mice where they proliferated upon FV-infection. Using intravital two-photon microscopy we visualized CTL motility in the bone marrow and evaluated cytotoxic molecule expression by flow cytometry. Knockout of PDCD1 improved the CTL motility at 14 days post infection and enhanced the expression of cytotoxicity markers. Our data show the potential of genetic tuning of naive antiviral CTLs and might be relevant for future designs of improved T cell-mediated therapies.

Autophagy degrades immunogenic endogenous retroelements induced by 5-azacytidine in acute myeloid leukemia

The hypomethylating agent 5-azacytidine (AZA) is the first-line treatment for AML patients unfit for intensive chemotherapy. The effect of AZA results in part from T-cell cytotoxic responses against MHC-I-associated peptides (MAPs) deriving from hypermethylated genomic regions such as cancer-testis antigens (CTAs), or endogenous retroelements (EREs). However, evidence supporting higher ERE MAPs presentation after AZA treatment is lacking. Therefore, using proteogenomics, we examined the impact of AZA on the repertoire of MAPs and their source transcripts. AZA-treated AML upregulated both CTA and ERE transcripts, but only CTA MAPs were presented at greater levels. Upregulated ERE transcripts triggered innate immune responses against double-stranded RNAs but were degraded by autophagy, and not processed into MAPs. Autophagy resulted from the formation of protein aggregates caused by AZA-dependent inhibition of DNMT2. Autophagy inhibition had an additive effect with AZA on AML cell proliferation and survival, increased ERE levels, increased pro-inflammatory responses, and generated immunogenic tumor-specific ERE-derived MAPs. Finally, autophagy was associated with a lower abundance of CD8+ T-cell markers in AML patients expressing high levels of EREs. This work demonstrates that AZA-induced EREs are degraded by autophagy and shows that inhibiting autophagy can improve the immune recognition of AML blasts in treated patients.

Efficacy of LCMV-based cancer immunotherapies is unleashed by intratumoral injections of polyI:C

BackgroundLymphocytic choriomeningitis virus (LCMV) belongs to the Arenavirus family known for inducing strong cytotoxic T-cell responses in both mice and humans. LCMV has been engineered for the development of cancer...

In Situ Hydrogel ModulatescDC1-Based Antigen Presentation and Cancer Stemness to Enhance Cancer Vaccine Efficiency.

Effective presentation of antigens by dendritic cells (DC) is essential for achieving a robust cytotoxic T lymphocytes (CTLs) response, in which cDC1 is the key DC subtype for high-performance activation of CTLs. However, low cDC1 proportion, complex process, and high cost severely hindered cDC1 generation and application. Herein, the study proposes an in situ cDC1 recruitment and activation strategy with simultaneous inhibiting cancer stemness for inducing robust CTL responses and enhancing the anti-tumor effect. Fms-like tyrosine kinase 3 ligand (FLT3L), Poly I:C, and Nap-CUM (NCUM), playing the role of cDC1 recruitment, cDC1 activation, inducing antigen release and decreasing tumor cell stemness, respectively, are co-encapsulated in an in situ hydrogel vaccine (FP/NCUM-Gel). FP/NCUM-Gel is gelated in situ after intra-tumoral injection. With the near-infrared irradiation, tumor cell immunogenic cell death occurred, tumor antigens and immunogenic signals are released in situ. cDC1 is recruited to tumor tissue and activated for antigen cross-presentation, followed by migrating to lymph nodes and activating CTLs. Furthermore, tumor cell stemness are inhibited by napabucasin, which can help CTLs to achieve comprehensive tumor killing. Collectively, the proposed strategy of cDC1 in situ recruitment and activation combined with stemness inhibition provides great immune response and anti-tumor potential, providing new ideas for clinical tumor vaccine design.

Abstract 3965: A cytolytic effector CD8+ T cell response is associated with remission in adult T-cell leukemia/lymphoma

Abstract Introduction: Adult T-cell leukemia/lymphoma (ATLL) is a rare blood cancer that develops in 3-5% of human T-lymphotropic virus-1 (HTLV1) carriers when the combination of viral oncoproteins and somatic mutations lead to malignant transformation of HTLV1 infected CD4+ T cells. ATLL has the worst overall survival among peripheral T cell lymphomas and remains a fatal disease despite efforts to improve outcomes over the last 35 years. First-line combination chemotherapy rarely achieves a durable response and two immune-modulating treatments (α-CCR4 and α-PD-1 monoclonal antibodies) failed in clinical trials. These findings highlight an urgent need to improve our understanding of the functional state of host immunity in ATLL patients. A host immune response against HTLV1 is pivotal in preventing ATLL development. Malignant ATLL cells can escape immune surveillance by preventing antigen presentation and engaging immune checkpoints. The prospect of an ATLL clone inducing systemic suppression was suggested in the literature but has not been directly examined. Furthermore, HTLV1-specific CD8+ cytotoxic T lymphocytes (CTLs) play an important role during viral latency, yet it remains unknown if they play a clinically relevant role during ATLL treatment. Methodology: In this work, we hypothesized that malignant ATLL cells will exhibit a suppressive immune phenotype and that effective therapeutic interventions will augment anti-ATLL CTLs to promote durable remission. We implemented an immune profiling approach to analyze PBMC samples from ATLL patients. Specifically, we developed a powerful 30-marker spectral flow panel to discriminate malignant ATLL cells from non-transformed lymphocytes (T, B, and NK cells) and monitor the expression of key transcription factors and markers associated with proliferation, cytolysis, and exhaustion. We analyzed diagnostic samples from the Montefiore-Einstein ATLL Biobank and samples collected during a phase 2 clinical trial aimed to assess the efficacy of a promising Belinostat/Zidovudine/Interferon-α combination therapy against ATLL (NCT02737046). Results: Our findings reveal that malignant ATLL cells exhibit an immune phenotype associated with robust immunosuppression and yet distinct from other suppressive immune cells (i.e., regulatory T cells). Additionally, we discovered that ATLL patient samples contained a unique population of phenotypically exhausted CD8+ T cells, the proportion of which was drastically reduced in patients under remission. Remarkably, a decrease in this exhausted subset correlated with the expansion of highly cytolytic CD8+ CTLs. Conclusion: Altogether, our findings suggest that ATLL cells may drive CD8+ CTLs into exhaustion to achieve immune evasion and that a robust anti-ATLL CTL response actively contributes to a remission state. Citation Format: Erik Guillen, Eric Liu, Yanhua Wang, Aditi Shastri, Alejandro Sica, Amit Verma, Xingxing Zhang, Juan Carlos Ramos, Hilda Ye, Gregoire Lauvau. A cytolytic effector CD8+ T cell response is associated with remission in adult T-cell leukemia/lymphoma [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 3965.

Targeting Epstein-Barr virus in multiple sclerosis: when and how?

Epidemiological evidence implicates Epstein-Barr virus (EBV) as the cause of multiple sclerosis (MS). However, its biological role in the pathogenesis of MS is uncertain. The article provides an overview of the role of EBV in the pathogenesis of MS and makes a case for targeting EBV as a treatment strategy for MS. EBV potentially triggers autoimmunity via molecular mimicry or immune dysregulation. Another hypothesis, supported by immunological and virological data, indicates that active EBV infection via latent-lytic infection cycling within the central nervous system or periphery drives MS disease activity. This supports testing small molecule anti-EBV agents targeting both latent and lytic infection, central nervous system-penetrant B-cell therapies and EBV-targeted immunotherapies in MS. Immunotherapies may include EBV-specific cytotoxic or chimeric antigen receptors T-cells, therapeutic EBV vaccines and immune reconstitution therapies to boost endogenous EBV-targeted cytotoxic T-cell responses. EBV is the probable cause of MS and is likely to be driving MS disease activity via latent-lytic infection cycling. There is evidence that all licensed MS disease-modifying therapies target EBV, and there is a compelling case for testing other anti-EBV strategies as potential treatments for MS.

Abstract 4105: Immune-modulating vaccines targeting CD73

Abstract Background: To realize the generalizable regulation of the tumor microenvironment (TME), immune-modulating vaccines are developed to target shared antigens expressed by immunosuppressive cells and cancer cells. CD73, also known as ecto-5'-nucleotidase, is a pivotal enzyme in the adenosine metabolic pathway and is expressed on the surface of multiple cancer cells and immunosuppressive cells in the TME. In this study, we aim to develop a CD73-targeting immune modulatory peptide vaccine by predicting antigen sequences, screening immunogenic antigen peptides, and assessing their anti-tumor efficacy and capacity to improve the TME. Methods: The expression level of CD73 on various cancer cell lines were evaluated by flow cytometry. Several online software were used to predict CD73-derived peptides that bind to the mouse MHC-I (major histocompatibility complex class I) molecules. Cells from the draining lymph nodes of healthy mice and peptide-immunized mice were harvested and stimulated by the synthetic peptides in vitro. T cell activation level, IFN-γrelease degree and their cytolytic capability towards cancer cells was measured. CD73-derived peptides were mixed with CpG to treat 4T1 tumor-bearing mice and the changes in the TME were investigated by flow cytometry. The online website (www.camoip.net) was used to analyze the correlation between CD73 expression and patient survival in the TCGA Breast invasive carcinoma (BRCA) cohort.Results: 4T1 exhibited a strikingly elevated surface expression of CD73 compared to other mouse cancer cell lines. Three CD73-derived peptides (EP1, EP2, EP3) consisting of 8-10 amino acids were synthesized. In vitro stimulation experiments indicated that EP1 and EP3 significantly elicited T-cell responses in draining lymph nodes. After stimulation, the proportions of CD25+, CD69+, CD107a+, and CD137+ cells markedly increased in CD8+ T cell populations. These cells also displayed enhanced cytotoxicity against 4T1 tumor cells. In vivo experiments demonstrated that EP3 induced a pronounced anti-tumor response in an established 4T1 tumor model without evident toxicity. Moreover, subcutaneous injection of EP3 improves the TME by promoting antigen-specific CD8+ T-cell responses, reducing M2 macrophages, and inducing neutrophil maturation. High tumor CD73 expression was also found to be associated with poor outcomes among BRCA patients, which further indicated clinical relevance. Conclusions: In this study, we successfully identified an immunogenic vaccine targeting CD73. It expressed dual functionality by effectively triggering cytotoxic T-cell responses that directly killing CD73-highexpressing tumor cells and regulating the tumor microenvironment through eliminating immunosuppressive cells with elevated CD73 expression. These findings hold promise for a novel immune-modulating vaccine and suggest a potential therapeutic avenue for the treatment of breast cancer and other types of cancer. Citation Format: Yuting Luo, Tao Shi, Hanbing Wang, Baorui Liu, Jia Wei. Immune-modulating vaccines targeting CD73 [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 4105.

Abstract 3249: Simple, rapid, and robust bioluminescent cell-based assay for detecting neutralizing antibodies against AAV in serum

Abstract Adeno-associated viruses (AAV) are prevalent gene therapy vectors, delivering therapeutic transgenes to treat various diseases including cancer and rare monogenic diseases. Individuals with pre-existing immunity to AAVs are less likely to benefit from AAV-based therapies due to both, a reduction in cellular AAV uptake and activation of a cytotoxic T-cell response to transduced cells, decreasing efficacy or changing the safety profile. Measurement of serum neutralizing antibodies (NAbs) against AAV is essential to determine treatment eligibility and to identify clinical trial candidates. Here we introduce a cell-based assay using NanoLuc® luciferase to detect NAbs in serum samples. The superior brightness of NanoLuc® luciferase in our AAV reporter system results in the need of low concentrations of AAV and a short 24h assay time. The minimal AAV concentration in our assay aids in detecting low NAb titers in serum samples, reducing false negatives and enhancing decision-making accuracy. Testing 60 human serum samples against a range of AAV serotypes, we found that a large fraction of the population had pre-existing NAbs to at least one serotype, and of the pre-exposed individuals a majority displayed NAbs against multiple AAV serotypes. Interestingly, fewer samples were seropositive for AAV-DJ than for either parental serotype (AAV2, AAV8 or AAV9), highlighting the assays utility in the development of engineered capsids. Furthermore, the broad dynamic range of our assay facilitates serum sample categorization into four NAb tiers: negative, low, medium, and high using a single serum dilution. Neutralizing titers (ND50) which was determined by serial dilution of the serum samples strongly agreed with the assigned NAb tiers. Our study with 40 naïve mice further validated the assay's robustness, showing a clear correlation between NAb levels and injected AAV viral loads. Taken together, this rapid, highly sensitive, and reliable assay enabled by NanoLuc® AAV reporter technology precisely measures NAbs against AAVs in both human and animal serum. Further validation of the assay is required for future use in a clinical setting. Citation Format: Morten Seirup, Kirsten Wingate, Jeff Nelson, Kara Machleidt, Vanessa Ott, Trish Hoang, Marjeta Urh. Simple, rapid, and robust bioluminescent cell-based assay for detecting neutralizing antibodies against AAV in serum [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 3249.

Adenosine Increases the Immunosuppressive Capacity of Cervical Cancer Cells by Increasing PD-L1 Expression and TGF-β Production through Its Interaction with A2AR/A2BR.

The present study provides evidence showing that adenosine (Ado) increases the expression of programmed death ligand 1 (PD-L1) in cervical cancer (CeCa) cells by interacting with A2AR/A2BR and that TGF-β1 acts in an autocrine manner to induce PD-L1 expression, enhancing the immunosuppressive effects of CeCa cells on activated T lymphocytes (ATLs) and CD8+ cytotoxic T lymphocytes (CTLs) specific for antigenic peptides derived from E6 and E7 proteins of HPV-16. Interestingly, the addition of the antagonists ZM241385 and MRS1754, which are specific for A2AR and A2BR, respectively, or SB-505124, which is a selective TGF-β1 receptor inhibitor, to CeCa cell cultures significantly inhibited PD-L1 expression. In addition, supernatants from CeCa cells that were treated with Ado (CeCa-Ado Sup) increased the expression of PD-1, TGF-β1, and IL-10 and decreased the expression of IFN-γ in ATLs. Interestingly, the addition of an anti-TGF-β neutralizing antibody strongly reversed the effect of CeCa-Ado Sup on PD-1 expression in ATLs. These results strongly suggest the presence of a feedback mechanism that involves the adenosinergic pathway, the production of TGF-β1, and the upregulation of PD-L1 expression in CeCa cells that suppresses the antitumor response of CTLs. The findings of this study suggest that this pathway may be clinically important and may be a therapeutic target.

Viral infection dynamics with immune chemokines and CTL mobility modulated by the infected cell density.

We study a viral infection model incorporating both cell-to-cell infection and immune chemokines. Based on experimental results in the literature, we make a standing assumption that the cytotoxic T lymphocytes (CTL) will move toward the location with more infected cells, while the diffusion rate of CTL is a decreasing function of the density of infected cells. We first establish the global existence and ultimate boundedness of the solution via a priori energy estimates. We then define the basic reproduction number of viral infection and prove (by the uniform persistence theory, Lyapunov function technique and LaSalle invariance principle) that the infection-free steady state is globally asymptotically stable if . When , then becomes unstable, and another basic reproduction number of CTL response becomes the dynamic threshold in the sense that if , then the CTL-inactivated steady state is globally asymptotically stable; and if , then the immune response is uniform persistent and, under an additional technical condition the CTL-activated steady state is globally asymptotically stable. To establish the global stability results, we need to prove point dissipativity, obtain uniform persistence, construct suitable Lyapunov functions, and apply the LaSalle invariance principle.

CD4+ T cells produce IFN-I to license cDC1s for induction of cytotoxic T-cell activity in human tumors

CD4+ T cells can "help” or "license” conventional type 1 dendritic cells (cDC1s) to induce CD8+ cytotoxic T lymphocyte (CTL) anticancer responses, as proven in mouse models. We recently identified cDC1s with a transcriptomic imprint of CD4+ T-cell help, specifically in T-cell-infiltrated human cancers, and these cells were associated with a good prognosis and response to PD-1-targeting immunotherapy. Here, we delineate the mechanism of cDC1 licensing by CD4+ T cells in humans. Activated CD4+ T cells produce IFNβ via the STING pathway, which promotes MHC-I antigen (cross-)presentation by cDC1s and thereby improves their ability to induce CTL anticancer responses. In cooperation with CD40 ligand (L), IFNβ also optimizes the costimulatory and other functions of cDC1s required for CTL response induction. IFN-I-producing CD4+ T cells are present in diverse T-cell-infiltrated cancers and likely deliver “help” signals to CTLs locally, according to their transcriptomic profile and colocalization with “helped/licensed” cDCs and tumor-reactive CD8+ T cells. In agreement with this scenario, the presence of IFN-I-producing CD4+ T cells in the TME is associated with overall survival and the response to PD-1 checkpoint blockade in cancer patients.

PD-1 or CTLA-4 blockade promotes CD86-driven Treg responses upon radiotherapy of lymphocyte-depleted cancer in mice.

Radiotherapy (RT) is considered immunogenic, but clinical data demonstrating RT-induced T cell priming are scarce. Here, we show in a mouse tumor model representative of human lymphocyte-depleted cancer that RT enhanced spontaneous priming of thymus-derived (FOXP3+Helios+) Tregs by the tumor. These Tregs acquired an effector phenotype, populated the tumor, and impeded tumor control by a simultaneous, RT-induced CD8+ cytotoxic T cell (CTL) response. Combination of RT with CTLA-4 or PD-1 blockade, which enables CD28 costimulation, further increased this Treg response and failed to improve tumor control. We discovered that upon RT, the CD28 ligands CD86 and CD80 differentially affected the Treg response. CD86, but not CD80, blockade prevented the effector Treg response, enriched the tumor-draining lymph node migratory conventional DCs that were positive for PD-L1 and CD80 (PD-L1+CD80+), and promoted CTL priming. Blockade of CD86 alone or in combination with PD-1 enhanced intratumoral CTL accumulation, and the combination significantly increased RT-induced tumor regression and OS. We advise that combining RT with PD-1 and/or CTLA-4 blockade may be counterproductive in lymphocyte-depleted cancers, since these interventions drive Treg responses in this context. However, combining RT with CD86 blockade may promote the control of such tumors by enabling a CTL response.

Intratumoral delivery of a highly active form of XCL1 enhances antitumor CTL responses through recruitment of CXCL9-expressing conventional type-1 dendritic cells.

Conventional type 1 dendritic cells (cDC1s) play a crucial role in antitumor immunity through the induction and activation of tumor-specific CD8+ cytotoxic T cells (CTLs). The chemokine XCL1 is a major chemotactic factor for cDC1s and its receptor XCR1 is selectively expressed on cDC1s. Here, we investigated the effect of intratumoral delivery of a highly active form of murine XCL1 (mXCL1-V21C/A59C) on cDC1-mediated antitumor immunity using a hydrophilic gel patch. The hydrophilic gel patch containing mXCL1-V21C/A59C increased cDC1 accumulation in the tumor masses and promoted their migration to the regional lymph nodes, resulting in enhanced induction of tumor-specific CTLs. Tumor-infiltrating cDC1s not only expressed XCR1 but also produced CXCL9, a ligand for CXCR3 which is highly expressed on CTLs and NK cells. Consequently, CTLs and NK cells were increased in the tumor masses of mice treated with mXCL1-V21C/A59C, while immunosuppressive cells such as monocyte-derived suppressive cells and regulatory T cells were decreased. We also confirmed that anti-CXCL9 treatment decreased the tumor infiltration of CTLs. The intratumoral delivery of mXCL1-V21C/A59C significantly decreased tumor growth and prolonged survival in E.G7-OVA and B16-F10 tumor-bearing mice. Furthermore, the antitumor effect of mXCL1-V21CA59C was enhanced in combination with anti-programmed cell death protein 1 treatment. Finally, using The Cancer Genome Atlas database, we found that XCL1 expression was positively correlated with tumor-infiltrating cDC1s and a better prognosis in melanoma patients. Collectively, our findings provide a novel therapeutic approach to enhance tumor-specific CTL responses through the selective recruitment of CXCL9-expressing cDC1s into the tumor masses.

SMART-lipid nanoparticles enabled mRNA vaccine elicits cross-reactive humoral responses against the omicron sub-variants

The continual emergence of SARS-CoV-2 variants has necessitated the development of broad cross-reactive vaccines. Recent findings suggest that enhanced antigen presentation could lead to cross-reactive humoral responses against the emerging variants. Towards enhancing the antigen presentation to dendritic cells (DCs), we developed a novel shikimoylated mannose receptor targeting lipid nanoparticle system (SMART-LNPs) that could effectively deliver mRNAs into DCs. To improve the translation of mRNA, we developed spike domain-based trimeric-S1 (TS1) mRNA with optimized codon sequence, base modification, and engineered 5’&3’ UTRs. In a mouse model, SMART-LNPs-TS1 vaccine could elicit robust broad cross-reactive IgGs against Omicron sub-variants, and induced IFNγ producing T-cells against SARS-CoV-2 virus compared to non-targeted LNPs-TS1 vaccine. Further, T-cells analysis revealed that SMART-LNPs-TS1 vaccine induced long-lived memory T-cell subsets, Th1-dominant and cytotoxic T-cells immune responses against SARS-CoV-2 virus. Importantly, SMART-LNPs-TS1 vaccine produced strong Th1-predominant humoral and cellular immune responses. Overall, SMART-LNPs can be explored for precise antigenic mRNA delivery and robust immune responses. This platform technology can be explored further as a next-gen delivery system for mRNA-based immune therapies.

Global stability of secondary DENV infection models with non-specific and strain-specific CTLs

Dengue virus (DENV) is a highly perilous virus that is transmitted to humans through mosquito bites and causes dengue fever. Consequently, extensive efforts are being made to develop effective treatments and vaccines. Mathematical modeling plays a significant role in comprehending the dynamics of DENV within a host in the presence of cytotoxic T lymphocytes (CTL) immune response. This study examines two models for secondary DENV infections that elucidate the dynamics of DENV under the influence of two types of CTL responses, namely non-specific and strain-specific responses. The first model encompasses five compartments, which consist of uninfected monocytes, infected monocytes, free DENV particles, non-specific CTLs, and strain-specific CTLs. In the second model, latently infected cells are introduced into the model. We posit that the CTL responsiveness is determined by a combination of self-regulating CTL response and a predator-prey-like CTL response. The model's solutions are verified to be nonnegativity and bounded and the model possesses two equilibrium states: the uninfected equilibrium EQ0 and the infected equilibrium EQ⁎. Furthermore, we calculate the basic reproduction number R0, which determines the existence and stability of the model's equilibria. We examine the global stability by constructing suitable Lyapunov functions. Our analysis reveals that if R0≤1, then EQ0 is globally asymptotically stable (G.A.S), and if R0>1, then EQ0 is unstable while EQ⁎ is G.A.S. To illustrate our findings analytically, we conduct numerical simulations for each model. Additionally, we perform sensitivity analysis to demonstrate how the parameter values of the proposed model impact R0 given a set of data. Finally, we discuss the implications of including the CTL immune response and latently infected cells in the secondary DENV infection model. Our study demonstrates that incorporating the CTL immune response and latently infected cells diminishes R0 and enhances the system's stability around EQ0.

In vivo assembly of epitope-coated biopolymer particles that induce anti-tumor responses

There is an unmet need for antigen delivery systems that elicit efficient T cell priming to prevent infectious diseases or for treatment of cancers. Here, we explored the immunogenic potential of biologically assembled biopolymer particles (BPs) that have been bioengineered to display the antigenic MHC I and MHC II epitopes of model antigen ovalbumin (OVA). Purified dendritic cells (DCs) captured BP-OVA and presented the associated antigenic epitopes to CD4+ T cells and CD8+ T cells. Vaccination with BP-OVA in the absence of adjuvant elicited antigen presentation to OVA-specific CD8+ and CD4+ T cells and cross-primed effective cytotoxic T lymphocyte (CTL) killers. BP-OVA induction of CTL killing did not require CD4+ T cell help, with active CTLs generated in BP-OVA vaccinated I-Ab−/− and CD40−/− mice. In contrast, IL-15 and type I IFN were required, with abrogated CTL activity in vaccinated IL-15−/− and IFNAR1−/− mice. cDC1 and/or CD103+ DCs were not essential for BP-OVA specific CTL with immunization eliciting responses in Batf3−/− mice. Poly I:C, but not LPS or CpG, co-administered as an adjuvant with BP-OVA boosted CTL responses. Finally, vaccination with BP-OVA protected against B16-OVA melanoma and Eμ-myc-GFP-OVA lymphoma inoculation. In summary, we have demonstrated that epitope-displaying BPs represent an antigen delivery platform exhibiting a unique mechanism to effectively engage T cell immune responses.

C/EBPα mediates the maturation and antitumor functions of macrophages in human hepatocellular carcinoma

Recent studies have suggested that therapeutic upregulation of CCAAT/enhancer binding protein α (C/EBPα) prevents hepatocellular carcinoma (HCC) progression. However, the mechanisms underlying this outcome are not fully understood. In this study, we investigated the expression and functional roles of C/EBPα in human HCC, with a focus on monocytes/macrophages (Mφs). Paraffin-embedded tissues were used to visualize C/EBPα expression and analyze the prognostic value of C/EBPα+ monocytes/Mφs in HCC patients. The underlying regulatory mechanisms were examined using human monocyte-derived Mφs. The results showed that the expression of C/EBPα on monocytes/Mφs was significantly decreased in intra-tumor tissues compared to the corresponding peri-tumor tissues. C/EBPα+ monocytes/Mφs displayed well-differentiation and antitumor capacities, and the accumulation of these cells in tissue was associated with antitumor immune responses and predicted longer overall survival (OS) of HCC patients. Mechanistic studies demonstrated that C/EBPα was required for Mφ maturation and HLA-DR, CD169 and CD86 expression, which initiates antitumor cytotoxic T-cell responses; however, these effects were inhibited by monocyte autocrine IL-6- and IL-1β-induced suppression of mTOR1 signaling. Reprogramming Mφs via the upregulation of C/EBPα may provide a novel strategy for cancer immunotherapy in patients with HCC.

Exosome co-delivery of a STING agonist augments immunogenicity elicited by CVB3 VP1 vaccine via promoting antigen cross-presentation of CD8 + DCs

The induction of a robust CD8+ T cell response is critical for the success of an antiviral vaccine. In this study, we incorporated a STING agonist (SA) 2′3’-cGAMP into a previously developed exosome-based CVB3 viral myocarditis vaccine (Exo-VP1) to enhance its ability to induce CD8+ T cell responses and immunoprotection. Our results showed that compared to free SA adjuvant, exosome-mediated co-delivery (ExoSA-VP1) significantly enhanced SA uptake by dendritic cells (DCs) and more potently stimulated DC maturation. Immunization of mice showed that the ExoSA-VP1 vaccine-induced higher levels of CVB3-specific T cell proliferation and cytotoxicity, significantly increased the percentage of IFN-γ+CD8+ rather than CD4+ T cells, effectively reduced cardiac viral loads, attenuated myocarditis and improved survival in mice compared to the previous Exo-VP1 vaccine. Further investigation showed that ExoSA-VP1 significantly increased both the percentage and antigen cross-presentation capacity of splenic CD8+ DCs. Depletion of these CD8+ DCs by cytochrome C administration nearly abolished the advantage of ExoSA-VP1 in dominantly inducing IFN-γ+CD8+ cytotoxic T lymphocyte (CTL) production in immunized mice. Taken together, our results demonstrated the potential of ExoSA-VP1 as a promising candidate for anti-CVB3 vaccines and provide insights into immune-enhancing strategies aiming at augmenting antigen cross-presentation by DCs and enhancing potent CTL responses.

NF9 peptide specific cytotoxic T lymphocyte clone cross react to Y453F mutation of SARS-CoV-2 virus spike protein

The recognition by cytotoxic T cells (CTLs) is essential for the clearance of SARS-CoV-2 virus-infected cells. Several viral proteins have been described to be recognized by CTLs. Among them, the spike (S) protein is one of the immunogenic proteins. The S protein acts as a ligand for its receptors, and several mutants with different affinities for its cognate receptors have been reported, and certain mutations in the S protein, such as L452R and Y453F, have been found to inhibit the HLA-A24-restricted CTL response. In this study, we conducted a screening of candidate peptides derived from the S protein, specifically targeting those carrying the HLA-A24 binding motif. Among these peptides, we discovered that NF9 (NYNYLYRLF) represents an immunogenic epitope. CTL clones specific to the NF9 peptide were successfully established. These CTL clones exhibited the ability to recognize endogenously expressed NF9 peptide. Interestingly, the CTL clone demonstrated cross-reactivity with the Y453F peptide (NYNYLFRLF) but not with the L452R peptide (NYNYRYRLF). The CTL clone was able to identify the endogenously expressed Y453F mutant peptide. These findings imply that the NF9-specific CTL clone possesses the capability to recognize and respond to the Y453F mutant peptide.

Global Stability of a Delayed Model for the Interaction of SARS-CoV-2/ACE2 and Adaptive Immunity

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the culprit behind the coronavirus disease 2019 (COVID-19), which has killed millions of people. SARS-CoV-2 binds its spike (S) protein to the angiotensin-converting enzyme 2 (ACE2) receptor to inter the epithelial cells in the respiratory tracts. ACE2 is a crucial mediator in the SARS-CoV-2 infection pathway. In this paper, we construct a mathematical model to describe the SARS-CoV-2/ACE2 interaction and the adaptive immunological response. The model predicts the effects of latently infected cells as well as immunological responses from cytotoxic T lymphocytes (CTLs) and antibodies. The model is incorporated with three distributed time delays: (i) delay in the formation of latently infected epithelial cells, (ii) delay in the activation of latently infected epithelial cells, (iii) delay in the maturation of new released SARS-CoV-2 virions. We show that the model is well-posed and it admits five equilibria. The stability and existence of the equilibria are precisely controlled by four threshold parameters Ri, i=0,1,2,3. By formulating suitable Lyapunov functions and applying LaSalle's invariance principle, we show the global asymptotic stability for all equilibria. To demonstrate the theoretical results, we conduct numerical simulations. We do sensitivity analysis and identify the most sensitive parameters. We look at how the latent phase, ACE2 receptors, antibody and CTL responses, time delays affect the dynamical behavior of SARS-CoV-2. Although the basic reproduction number R0 is unaffected by the parameters of antibody and CTL responses, it is shown that viral replication can be hampered by immunological activation of antibody and CTL responses. Further, our findings indicate that R0 is affected by the rates at which the ACE2 receptor grows and degrades. This could provide valuable guidance for the development of receptor-targeted vaccines and medications. Furthermore, it is shown that, increasing time delays can effectively decrease R0 and then inhibit the SARS-CoV-2 replication. Finally, we show that, excluding the latently infected cells in the model would result in an overestimation of R0.