Mechanisms underlying response and resistance to immune checkpoint blockade in cancer immunotherapy.

Cancer immunotherapies targeting immune checkpoint pathways, such as programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), have achieved unprecedented therapeutic success in treating various types of cancer. The prominent and persistent clinical responses to immune checkpoint blockade (ICB) therapy are currently constrained to a subset of patients. Owing to discrete individual tumor and immune heterogeneity, most patients fail to benefit from ICB treatment, demonstrating either primary or acquired resistance. A thorough comprehension of the mechanisms restricting the efficacy of immune checkpoint inhibitors (ICIs) is required to extend their clinical applicability to a broader spectrum of patients and cancer types. Numerous studies are presently investigating potential prognostic markers of responsiveness, the complex dynamics underlying the therapeutic and adverse effects of ICB, and tumor immune evasion throughout the course of immunotherapy. In this article, we have reviewed the extant literature elucidating the mechanisms underlying the response and resistance to ICB, with a particular emphasis on PD-1 and CTLA-4 pathway blockade in the context of anti-tumor immunity. Furthermore, we aimed to explore potential approaches to overcome cancer therapeutic resistance and develop a rational design for more personalized ICB-based combinational regimens.

Knife's edge: Balancing immunogenicity and reactogenicity in mRNA vaccines.

Since the discovery of messenger RNA (mRNA), there have been tremendous efforts to wield them in the development of therapeutics and vaccines. During the COVID-19 pandemic, two mRNA vaccines were developed and approved in record-breaking time, revolutionizing the vaccine development landscape. Although first-generation COVID-19 mRNA vaccines have demonstrated over 90% efficacy, alongside strong immunogenicity in humoral and cell-mediated immune responses, their durability has lagged compared to long-lived vaccines, such as the yellow fever vaccine. Although worldwide vaccination campaigns have saved lives estimated in the tens of millions, side effects, ranging from mild reactogenicity to rare severe diseases, have been reported. This review provides an overview and mechanistic insights into immune responses and adverse effects documented primarily for COVID-19 mRNA vaccines. Furthermore, we discuss the perspectives of this promising vaccine platform and the challenges in balancing immunogenicity and adverse effects.

Galectin-4 increases the ability of M2 macrophages to enhance antiviral CD4+ T-cell responses.

Galectin-4 (Gal-4) is a ß-galactoside-binding protein belonging to the galectin family. Although Gal-4 is known to be involved in several physiologic processes of the gastrointestinal tract, its immunomodulatory roles remain unclear. In this study, we investigated whether Gal-4 influences the function of M1 and M2 macrophages. Gal-4 treatment drove more robust changes in the gene expression of M2 macrophages compared to M1 macrophages. Antiviral immune response-related genes were significantly upregulated in Gal-4-treated M2 macrophages. Gal-4 significantly enhanced the immunostimulatory activity of M2 macrophages upon Toll-like receptor 7 stimulation or infection with lymphocytic choriomeningitis virus (LCMV). Moreover, the antibody production against LCMV infection and the antiviral CD4+ T-cell responses, but not the antiviral CD8+ T-cell responses, were greatly increased by Gal-4-treated M2 macrophages in vivo. The present results indicate that Gal-4 enhances the ability of M2 macrophages to promote antiviral CD4+ T-cell responses. Thus, Gal-4 could be used to boost antiviral immune responses.

In planta Production and Validation of Neuraminidase Derived from Genotype 4 Reassortant Eurasian Avian-like H1N1 Virus as a Vaccine Candidate.

Influenza viruses are a major public health threat that causes repetitive outbreaks. In recent years, genotype 4 (G4) reassortant Eurasian avian-like (EA) H1N1 (G4 EA H1N1) has garnered attention as a potential novel pandemic strain. The necessity of developing vaccines against G4 EA H1N1 is growing because of the increasing cases of human infection and the low cross-reactivity of the strain with current immunity. In this study, we produced a G4 EA H1N1-derived neuraminidase (G4NA) as a vaccine candidate in Nicotiana benthamiana. The expressed G4NA was designed to be accumulated in the endoplasmic reticulum (ER). The M-domain of the human receptor-type tyrosine-protein phosphatase C was incorporated into the expression cassette to enhance the translation of G4NA. In addition, the family 3 cellulose-binding module and Brachypodium distachyon small ubiquitin-like modifier sequences were used to enable the cost-effective purification and removal of unnecessary domains after purification, respectively. The G4NA produced in plants displayed high solubility and assembled as a tetramer, which is required for the efficacy of an NA-based vaccine. In a mouse immunization model, the G4NA produced in plants could induce significant humoral immune responses. The plant-produced G4NA also stimulated antigen-specific CD4 T cell activation. These G4NA vaccine-induced immune responses were intensified by the administration of the antigen with a vaccine adjuvant. These results suggest that G4NA produced in plants has great potential as a vaccine candidate against G4 EA H1N1.

Immunogenicity and Durability of Antibody Responses to Homologous and Heterologous Vaccinations with BNT162b2 and ChAdOx1 Vaccines for COVID-19.

During the COVID-19 pandemic, vaccines were developed based on various platform technologies and were approved for emergency use. However, the comparative analysis of immunogenicity and durability of vaccine-induced antibody responses depending on vaccine platforms or vaccination regimens has not been thoroughly examined for mRNA- or viral vector-based vaccines. In this study, we assessed spike-binding IgG levels and neutralizing capacity in 66 vaccinated individuals prime-boost immunized either by homologous (BNT162b2-BNT162b2 or ChAdOx1-ChAdOx1) or heterologous (ChAdOx1-BNT162b2) vaccination for six months after the first vaccination. Despite the discrepancy in intervals for the prime-boost vaccination regimen of different COVID-19 vaccines, we found stronger induction and relatively rapid waning of antibody responses by homologous vaccination of the mRNA vaccine, while weaker boost effect and stable maintenance of humoral immune responses were observed in the viral vector vaccine group over 6 months. Heterologous vaccination with ChAdOx1 and BNT162b2 resulted in an effective boost effect with the highest remaining antibody responses at six months post-primary vaccination.

Anti-influenza A virus activity by Agrimonia pilosa and Galla rhois extract mixture.

Influenza A virus (IAV) continues to threaten human health. To date, two classes of antiviral drugs have been approved to treat IAV infection, but the continuous emergence of the drug-resistant IAV mutant reinforces the need to develop new antiviral drugs. In this study, we aimed to investigate the anti-IAV activity of an aqueous mixture of Agrimonia pilosa and Galla rhois extracts (APRG64). We demonstrated that APRG64 significantly reduced the IAV-induced cytopathic effect, the transcription/expression of viral proteins, and the production of infectious viral particles. Among nine major components of APRG64, apigenin was identified as the main ingredient responsible for the anti-IAV activity. Interestingly, APRG64 and apigenin inhibited the cell attachment and entry of virus and polymerase activity. Importantly, intranasal administration of APRG64 or apigenin strongly reduced viral loads in the lungs of IAV-infected mice. Furthermore, oral administration of APRG64 significantly reduced the level of viral RNAs and the expression level of pro-inflammatory cytokines in the lungs, which protected mice from IAV-induced mortality. In conclusion, APRG64 could be an attractive antiviral drug to treat IAV infection.

Potent antiviral activity of the extract of Elaeocarpus sylvestris against influenza A virus in vitro and in vivo.

BACKGROUND: Elaeocarpus sylvestris (Lour.) Poir. (Elaeocarpaceae) belongs to a genus of tropical and semitropical evergreen trees, which has known biological activities such as antiviral and immunomodulatory activities. However, its antiviral potential against influenza virus infection remains unknown. PURPOSE: In this study, we investigated the antiviral activity of the 50% aqueous ethanolic extract of E. sylvestris (ESE) against influenza A virus (IAV) infection, which could lead to the development of novel phytomedicine to treat influenza virus infection. METHODS: To investigate the in vitro antiviral activity of ESE and its main ingredients, 1,​2,​3,​4,​6-​penta-​O-​galloyl-ß-d-glucose (PGG) and geraniin (GE), the levels of viral RNAs, proteins, and infectious viral particles in IAV-infected MDCK cells were analyzed. Molecular docking analysis was performed to determine the binding energy of PGG and GE for IAV proteins. To investigate in vivo antiviral activity, IAV-infected mice were treated intranasally or intragastrically with ESE, PGG, or GE. RESULTS: ESE and its gallate main ingredients (PGG and GE) strongly inhibited the production of viral RNAs, viral proteins, and infectious viral particles in vitro. Also through the viral attachment on cells, polymerase activity, signaling pathway, we revealed the ESE, PGG, and GE inhibit multiple steps of IAV replication. Molecular docking analysis revealed that PGG and GE could interact with 12 key viral proteins (M1, NP, NS1 effector domain (ED), NS1 RNA-binding domain (RBD), HA pocket A, HA receptor-binding domain (RBD), NA, PA, PB1, PB2 C-terminal domain, PB2 middle domain, and PB2 cap-binding domain) of IAV proteins with stable binding energy. Furthermore, intranasal administration of ESE, PGG, or GE protected mice from IAV-induced mortality and morbidity. Importantly, oral administration of ESE suppressed IAV replication and the expression of inflammatory cytokines such as IFN-γ, TNF-α, and IL-6 in the lungs to a large extent. CONCLUSION: ESE and its major components (PGG and PE) exhibited strong antiviral activity in multiple steps against IAV infection in silico, in vivo, and in vitro. Therefore, ESE could be used as a novel natural product derived therapeutic agent to treat influenza virus infection.

Role of Damage-Associated Molecular Pattern/Cell Death Pathways in Vaccine-Induced Immunity.

Immune responses induced by natural infection and vaccination are known to be initiated by the recognition of microbial patterns by cognate receptors, since microbes and most vaccine components contain pathogen-associated molecular patterns. Recent discoveries on the roles of damage-associated molecular patterns (DAMPs) and cell death in immunogenicity have improved our understanding of the mechanism underlying vaccine-induced immunity. DAMPs are usually immunologically inert, but can transform into alarming signals to activate the resting immune system in response to pathogenic infection, cellular stress and death, or tissue damage. The activation of DAMPs and cell death pathways can trigger local inflammation, occasionally mediating adaptive immunity, including antibody- and cell-mediated immune responses. Emerging evidence indicates that the components of vaccines and adjuvants induce immunogenicity via the stimulation of DAMP/cell death pathways. Furthermore, strategies for targeting this pathway to enhance immunogenicity are being investigated actively. In this review, we describe various DAMPs and focus on the roles of DAMP/cell death pathways in the context of vaccines for infectious diseases and cancer.

The amphibian peptide Yodha is virucidal for Zika and dengue viruses.

Zika virus (ZIKV) has emerged as a serious health threat in the Americas and the Caribbean. ZIKV is transmitted by the bite of an infected mosquito, sexual contact, and blood transfusion. ZIKV can also be transmitted to the developing fetus in utero, in some cases resulting in spontaneous abortion, fetal brain abnormalities, and microcephaly. In adults, ZIKV infection has been correlated with Guillain-Barre syndrome. Despite the public health threat posed by ZIKV, neither a vaccine nor antiviral drugs for use in humans are currently available. We have identified an amphibian host defense peptide, Yodha, which has potent virucidal activity against ZIKV. It acts directly on the virus and destroys Zika virus particles within 5 min of exposure. The Yodha peptide was effective against the Asian, African, and South American Zika virus strains and has the potential to be developed as an antiviral therapeutic in the fight against Zika virus. The peptide was also effective against all four dengue virus serotypes. Thus, Yodha peptide could potentially be developed as a pan-therapeutic for Zika and dengue viruses.

Squalene emulsion-based vaccine adjuvants stimulate CD8 T cell, but not antibody responses, through a RIPK3-dependent pathway.

The squalene-based oil-in-water emulsion (SE) vaccine adjuvant MF59 has been administered to more than 100 million people in more than 30 countries, in both seasonal and pandemic influenza vaccines. Despite its wide use and efficacy, its mechanisms of action remain unclear. In this study we demonstrate that immunization of mice with MF59 or its mimetic AddaVax (AV) plus soluble antigen results in robust antigen-specific antibody and CD8 T cell responses in lymph nodes and non-lymphoid tissues. Immunization triggered rapid RIPK3-kinase dependent necroptosis in the lymph node which peaked at 6 hr, followed by a sequential wave of apoptosis. Immunization with alum plus antigen did not induce RIPK3-dependent signaling. RIPK3-dependent signaling induced by MF59 or AV was essential for cross-presentation of antigen to CD8 T cells by Batf3-dependent CD8+ DCs. Consistent with this, RIPK3 deficient or Batf3 deficient mice were impaired in their ability to mount adjuvant-enhanced CD8 T cell responses. However, CD8 T cell responses were unaffected in mice deficient in MLKL, a downstream mediator of necroptosis. Surprisingly, antibody responses were unaffected in RIPK3-kinase or Batf3 deficient mice. In contrast, antibody responses were impaired by in vivo administration of the pan-caspase inhibitor Z-VAD-FMK, but normal in caspase-1 deficient mice, suggesting a contribution from apoptotic caspases, in the induction of antibody responses. These results demonstrate that squalene emulsion-based vaccine adjuvants induce antigen-specific CD8 T cell and antibody responses, through RIPK3-dependent and-independent pathways, respectively.

Programmed Necrosis and Disease:We interrupt your regular programming to bring you necroinflammation.

Compared to the tidy and immunologically silent death during apoptosis, necrosis seems like a chaotic and unorganized demise. However, we now recognize that there is a method to its madness, as many forms of necrotic cell death are indeed programmed and function beyond lytic cell death to support homeostasis and immunity. Inherently more immunogenic than their apoptotic counterpart, programmed necrosis, such as necroptosis, pyroptosis, ferroptosis, and NETosis, releases inflammatory cytokines and danger-associated molecular patterns (DAMPs), skewing the milieu to a pro-inflammatory state. Moreover, impaired clearance of dead cells often leads to inflammation. Importantly, these pathways have all been implicated in inflammatory and autoimmune diseases, therefore careful understanding of their molecular mechanisms can have long lasting effects on how we interpret their role in disease and how we translate these mechanisms into therapy.

B Cell Competition for Restricted T Cell Help Suppresses Rare-Epitope Responses.

The immune system responds preferentially to particular antigenic-epitopes contained within complex immunogens, such as proteins or microbes. This poorly understood phenomenon, termed "immunodominance," remains an obstacle to achieving polyvalent immune responses against multiple antigenic-epitopes through vaccination. We observed profound suppression in the hapten-specific antibody response in mice immunized with hapten-protein conjugate, mixed with an excess of protein, relative to that in mice immunized with hapten-protein alone. The suppression was robust (100-fold and 10-fold with a 10- or 2-fold excess of protein, respectively), stable over a 6-log range in antigen dose, observed within 10 days of vaccination, and resistant to boosting and adjuvants. Furthermore, there were reduced frequencies of antigen-specific germinal-center B cells and long-lived bone-marrow plasma cells. The mechanism of this "antigen-competition" was mediated largely by early access to T-helper cells. These results offer mechanistic insights into B cell competition during an immune response and suggest vaccination strategies against HIV, influenza, and dengue.

Mcl-1 regulates effector and memory CD8 T-cell differentiation during acute viral infection.

Mcl-1, an anti-apoptotic member of Bcl-2 family maintains cell viability during clonal expansion of CD8 T cells, but the cell intrinsic role of Mcl-1 in contraction of effectors or the number of memory CD8 T cells is unknown. Mcl-1 levels decline during the contraction phase but rebound to high levels in memory CD8 T cells. Therefore, by overexpressing Mcl-1 in CD8 T cells we asked whether limiting levels of Mcl-1 promote contraction of effectors and constrain CD8 T-cell memory. Mcl-1 overexpression failed to affect CD8 T-cell expansion, contraction or the magnitude of CD8 T-cell memory. Strikingly, high Mcl-1 levels enhanced mTOR phosphorylation and augmented the differentiation of terminal effector cells and effector memory CD8 T cells to the detriment of poly-cytokine-producing central memory CD8 T cells. Taken together, these findings provided unexpected insights into the role of Mcl-1 in the differentiation of effector and memory CD8 T cells.

Bach2 regulates homeostasis of Foxp3+ regulatory T cells and protects against fatal lung disease in mice.

Variants of the Bach2 gene are linked to vitiligo, celiac disease, and type 1 diabetes, but the underlying immunological mechanisms are unknown. In this study, we demonstrate that Bach2 plays crucial roles in maintaining T cell quiescence and governing the differentiation, activation, and survival of Foxp3(+) regulatory T (Treg) cells. Bach2-deficient T cells display spontaneous activation and produce elevated levels of Th1/Th2-type cytokines. Without Bach2, Treg cells exhibit diminished Foxp3 expression, depleted numbers, hyperactivation, enhanced proliferation, and profound loss of competitive fitness in vivo. Mechanistically, reduced survival of Bach2-deficient Treg cells was associated with reduced Bcl-2 and Mcl-1 levels and elevated Bim/Bcl-2 ratio. Additionally, Bach2 deficiency induced selective loss of Helios(-)Foxp3(+) Treg cells and a Treg cell transcriptome skewed toward the Th1/Th2 effector program at the expense of the Treg program. In vitro experiments confirmed that Bach2: 1) is indispensable for TCR/TGF-ß-induced Foxp3 expression; and 2) mitigates aberrant differentiation of Treg cells by repression of the competing Gata3-driven Th2 effector program. Importantly, perturbations in the differentiation of induced Treg cells was linked to a fatal Th2-type chronic inflammatory lung disease in Bach2-deficient mice. Thus, Bach2 enforces T cell quiescence, promotes the development and survival of Treg lineage, restrains aberrant differentiation of Treg cells, and protects against immune-mediated diseases.

FoxO1 controls effector-to-memory transition and maintenance of functional CD8 T cell memory.

During a T cell response, naive CD8 T cells differentiate into effector cells. Subsequently, a subset of effector cells termed memory precursor effector cells further differentiates into functionally mature memory CD8 T cells. The transcriptional network underlying this carefully scripted process is not well understood. In this study, we report that the transcription factor FoxO1 plays an integral role in facilitating effector-to-memory transition and functional maturation of memory CD4 and CD8 T cells. We find that FoxO1 is not required for differentiation of effector cells, but in the absence of FoxO1, memory CD8 T cells displayed features of senescence and progressive attrition in polyfunctionality, which in turn led to impaired recall responses and poor protective immunity. These data suggest that FoxO1 is essential for maintenance of functional CD8 T cell memory and protective immunity. Under competing conditions in bone marrow chimeric mice, FoxO1 deficiency did not perturb clonal expansion or effector differentiation. Instead, FoxO1-deficient memory precursor effector cells failed to survive and form memory CD8 T cells. Mechanistically, FoxO1 deficiency perturbed the memory CD8 T cell transcriptome, characterized by pronounced alterations in the expression of genes that encode transcription factors (including Tcf7), effector molecules, cell cycle regulators, and proteins that regulate fatty acid, purine, and pyramidine metabolism and mitochondrial functions. We propose that FoxO1 is a key regulator that reprograms and steers the differentiation of effector cells to functionally competent memory cells. These findings have provided fundamental insights into the mechanisms that regulate the quality of CD8 T cell memory to intracellular pathogens.

Role of PI3K/Akt signaling in memory CD8 T cell differentiation.

The clonal expansion, differentiation into effectors and establishing an immunological memory are crucial components of the adaptive immune response. Following the initial encounter with a pathogen, clonal CD8 T cell expansion yields at least two distinct populations of effector cells, short-lived effector cells (SLECs) and memory precursor effector cells (MPECs). SLECs are the terminally differentiated cells, which play an active role in pathogen clearance and undergo apoptosis once the pathogen is eliminated. In contrast, MPECs persist and give rise to self-renewing memory cells. These memory CD8 T cells maintain a state of heightened alertness and are poised to rapidly respond and swiftly clear the pathogen upon antigen re-encounter. As one of the goals of vaccination is to induce the development of these memory CD8 T cells, understanding the cellular and molecular basis of memory cell differentiation is critical to rational vaccine design. It is clear that memory differentiation is complex and involves multiple interrelated signaling pathways. It is influenced by factors such as the strength and duration of antigen receptor signaling and concurrent exposure to cytokines. Several signaling pathways that influence T cell fate have been recently described, and many culminate in the differential expression of specific transcription factors. Unfortunately, the mechanisms underlying the coordination and confluence of these signaling pathways remain largely unknown. In this review, we will discuss the role of the phosphatidylinositol 3-kinase signaling pathway as a central signaling node, and the function of Akt as a rheostat in orchestrating the differentiation of memory CD8 T cells.

FOXO3 regulates the CD8 T cell response to a chronic viral infection.

Chronic infections with viruses such as hepatitis B virus, hepatitis C virus, and HIV constitute a major global public health problem. Studies of chronic viral infections in humans and mice show that persistent antigenic stimulation induces dysregulation of T cell responses; virus-specific T cells either undergo clonal deletion or lose their ability to display the full spectrum of effector functions, a condition termed functional exhaustion. The ability to generate and retain sufficient numbers of functionally competent T cells, therefore, becomes vitally important in controlling chronic viral infections. Our understanding of the mechanisms governing T cell homeostasis during chronic viral infections, however, is poor. The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway controls cell fate decisions in many cell types by modulating the activity of downstream effectors, including the FOXO family of transcription factors. We have observed dynamic, in vivo alterations in the phosphorylation levels of three key proteins (Akt, FOXO1/FOXO3 [FOXO1/3], and mammalian target of rapamycin [mTOR]) involved in this signaling cascade and have identified the transcription factor FOXO3 as a negative regulator of the magnitude and effector function of CD8 T cells during chronic lymphocytic choriomeningitis virus (LCMV) infection in mice. We report that ablation of FOXO3 in T cells reduced apoptosis, increased the abundance of polyfunctional virus-specific CD8 T cells, and improved viral control. Thus, FOXO3 is a promising candidate target for immunotherapies of chronic viral infection.