Sustained Immune Response Research Articles

Toll‐like receptor 2 activation induces C–C chemokine receptor 2‐dependent natural killer cell recruitment to the peritoneum

Natural killer (NK) cells are innate effector cells with critical roles not only in tumor immunosurveillance and viral immunity, but also in bacterial and fungal infections. Toll‐like receptor 2 (TLR2) can be important in the early and sustained immune responses to pathogens and tumors through the induction of cytokines and chemokines that recruit and activate immune effector cells. We investigated the role of TLR2 activation in NK cell recruitment with a view to informing approaches to induce or regulate peritoneal NK cell responses therapeutically. Peritoneal injection of TLR2 activators, including peptidoglycan and the lipopeptides FSL‐1 and Pam3CSK4, resulted in NK cell recruitment after 16 h with increased NK cell numbers maintained for 48 h. TLR2 activators induced large amounts of CCR2 ligands, but much smaller amounts of CCR5 and CXCR3 ligands. Consistent with this observation, NK cell migration was abrogated in CCR2‐deficient mice after peritoneal FSL‐1 injection. Adoptive transfer of CCR2‐deficient NK cells prior to peritoneal FSL‐1 activation confirmed a cell‐intrinsic component of CCR2‐mediated NK cell migration. TLR2 activation did not induce an activated NK cell phenotype, but significant changes included an increase in the KLRG1+ subset and decreased NKG2D expression. Although not activated in vivo, peritoneal NK cells could be activated by interleukin (IL)‐12 and IL‐18 ex vivo to express CD69 and interferonγ. These data demonstrate that TLR2‐mediated immune activation is a potent inducer of NK cell recruitment via a CCR2‐dependent mechanism and that NK cells recruited by this mechanism can respond to additional signals to exert effector cell functions.

Coordinated and sustained immune memory responses after mild COVID-19.

Rodda, L. B. et al. Functional SARS-CoV-2-specific immune memory persists after mild COVID-19. Preprint at medRxiv https://doi.org/10.1101/2020.08.11.20171843 (2020) Article PubMed PubMed Central Google Scholar Download references OxImmuno Literature Initiative, University of Oxford, Oxford, UK Aljawharah Alrubayyi You can also search for this author in PubMed Google Scholar Correspondence to Aljawharah Alrubayyi. The author declares no competing interests. Reprints and Permissions Alrubayyi, A. Coordinated and sustained immune memory responses after mild COVID-19. Nat Rev Immunol (2020). https://doi.org/10.1038/s41577-020-00450-6 Download citation Published: 07 September 2020 DOI: https://doi.org/10.1038/s41577-020-00450-6

Fundamentals to clinical application of nanoparticles in cancer immunotherapy and radiotherapy

Cancer immunotherapy has made rapid progress over the past decade leading to high enthusiasm and interest worldwide. Codelivery of immunomodulators with chemotherapeutic agents and radioisotopes has been shown to elicit a strong and sustained immune response in animal models. Despite showing promising results in metastatic and recurrent cancers, the utilisation of immunotherapy in clinical settings has been limited owing to uncertainties in elicited immune response and occurrence of immune-related adverse events. These uncertainties can be overcome with the help of nanoparticles possessing unique properties for the effective delivery of targeted agents to specific sites. Nanoparticles play a crucial role in the effective delivery of cancer antigens and adjuvants, modulation of tumour microenvironment, production of long-term immune response and development of cancer vaccines. Here, we provide a comprehensive summary of nanotechnology-based cancer immunotherapy and radiotherapy including basics of nanotechnology, properties of nanoparticles and various methods of employing nanoparticles in cancer treatment. Thus, nanotechnology is anticipated to overcome the limitations of existing cancer immunotherapy and to effectively combine various cancer treatment modalities.

The second coming: The comeback of the live vaccines.

Live vaccines are gradually replaced by protein-based vaccines given the latter's better safety. But live vaccines seem to be more efficient via stimulation of the innate immune system.

Abstract 5393: Association of heat shock proteins as chaperone for STING: A potential link in a key immune activation mechanism revealed by the novel anti-cancer agent PV-10

Abstract Introduction: The activation of stimulator of interferon (IFN) genes (STING), an intracellular receptor system located in the endoplasmic reticulum, has been shown to augment antitumor immunity through the induction of pro-inflammatory cytokines. Currently, a number of STING agonists have been developed to treat refractory malignancies. Our previous studies have identified PV-10 (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein) as a novel therapeutic agent with potent activity following intra-tumoral injection. Here we describe a previously unidentified mechanism by which PV-10 may facilitate sustained immune activation and therapeutic antitumor activity. Methods: The well-established acute monocytic leukemia (AML) cell line THP-1 was used as a model to study STING activation in vitro. Cells were treated with PV-10 and the induction of STING was evaluated by Western blot analysis using cGAMP as a positive control. Proteins that associate with STING in the presence of PV-10 were purified by immunoprecipitation and analyzed by mass spectrometry (LC-MS/MS, Mascot database). The culture supernatants from PV-10 treated cells were probed for a panel of 42 immune cytokines using the Bio-Plex multiplex bead-based assay system. Results: We show that the exposure of THP-1 cells to PV-10 leads to the appearance of a new 70-KD STING dimer band detected by specific antibodies. Compared to cGAMP controls no induction of PDL-1 was noted. Mass spectrometric analysis of immuno-precipitates of STING in these cells showed the presence of Heat Shock Proteins (HSP) 60, 70 and 90 as well as Polyadenylate Binding Protein 1 (PABP1) to the dimerized STING complex. The chemokine assays showed specific upregulation of a distinct set of pro-inflammatory and cytotoxic T-cell recruitment cytokines. A peak in the induction of MCP-3 and IFN gamma was seen at 24 hours (2 fold) and an approximately 10-fold increase in IL-6, IL-8 and IP-10 was seen in the 24 hours following exposure to PV-10. A significant increase in MCP-1 levels was also noted. Discussion: The compound PV-10 has been shown to induce tumor necrosis following intra-tumoral injection. Our present data show a possible mechanism of this agent through STING dimerization and HSP association leading to a sustained pro-inflammatory and immune response. We provide experimental data, for the first time, for a role of HSPs in STING-mediated immune activation pathways and the description of an agent that may play a role in effective single-agent immunotherapy or drug combination therapy approaches in future clinical studies. Citation Format: Satbir Thakur, Chunfen Zhang, Laurent Brechenmacher, Luis Murgia-Favela, Aru Narendran. Association of heat shock proteins as chaperone for STING: A potential link in a key immune activation mechanism revealed by the novel anti-cancer agent PV-10 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5393.

Dendritic cells vaccines: Immunotherapy against cancer

Dendritic cells are specialized cells of the innate immune system, with high capacity to present antigens in the context of the Major Histocompatibility Complex II (MHC-II) to T lymphocytes (CD4+); these cells are up to 100 times stronger than any other antigen presenting cell. The ability of the antigen presentation by dendritic cells has been documented in animal models and clinical studies conducted in humans. Based on the above, different techniques and methods have been developed to use dendritic cells in cancer-aimed immunotherapies. The dendritic cell vaccines refer to biological therapies, prepared by different strategies (ex vivo and in vivo), which aim to enhance the presentation of tumor antigens and develop a more targeted and sustained immune response on these. They are obtained from precursor cells that mature with specific stimuli that direct them to the desired therapy. Different applications for these therapies have been described in numerous types of cancers, which will be described.

Sustained immune activation is associated with susceptibility to the amphibian chytrid fungus.

The disease chytridiomycosis caused by the fungus Bd has devastated amphibian populations worldwide. Functional genomic contributions to host susceptibility remain enigmatic and vary between species and populations. We conducted experimental Bd infections in Rana yavapaiensis, a species with intraspecific variation in chytridiomycosis susceptibility, to assess the skin and spleen transcriptomic response to infection over time. We predicted that increased immune gene expression would be associated with a positive disease outcome, but we instead found that surviving frogs had significantly reduced immune gene expression compared to susceptible frogs and to uninfected controls. MHC class IIβ gene expression was also significantly higher in susceptible frogs compared to surviving frogs. Furthermore, susceptible frogs expressed a significantly larger number of distinct class IIβ alleles, demonstrating a negative correlation between class IIβ expression, functional diversity, and survival. Expression of the MHC class IIβ locus previously associated with Bd disease outcomes was a significant predictor of Bd infection intensity at early infection stages but not at late infection stages, suggesting initial MHC-linked immune processes are important for ultimate disease outcomes. We infer through disease association and phylogenetic analysis that certain MHC variants are linked to the immune expression that was negatively associated with survival, and we hypothesize that frogs that did not express these alleles could better survive infections. Our study finds that MHC expression at early and late infection stages predicts Bd infection intensity, and suggests that generating a sustained immune response against Bd may be counterproductive for surviving chytridiomycosis in this partially susceptible species.

A novel rabies vaccine based on infectious propagating particles derived from hybrid VEEV-Rabies replicon.

BackgroundLive attenuated vaccines (LAVs) can mimic natural infection and have advantages to stimulate a robust and sustained immune response as well as to confer long-term protection. However, safety concerns is one of the major obstacles for LAVs development. In an effort to achieve the optimal balance between immunogenicity and safety, researchers currently have taken different strategies for the development of LAVs.MethodsWe constructed a novel infectious self-propagating hybrid replicon particle (PRP), VEEV-RABV-G, through replacing the entire structural proteins of the Venezuelan equine encephalitis virus (VEEV) with the glycoprotein of rabies virus (RABV-G) as the single structural protein. We evaluated the potential of VEEV-RABV-G as a safe live attenuated vaccine in mice model.FindingsWe found that VEEV-RABV-G could self-propagate efficiently in cell culture and induce a robust humoral immunity and provide protection against virulent RABV challenge in immunized mice. Remarkably, VEEV-RABV-G is highly attenuated in both adult and sucking mice, causing much weaker inflammatory and apoptotic effects in the brains of infected adult mice and significantly lower weight loss and morbidity compared with the commonly used RABV-derived LAVs.InterpretationThis study reveals the feasibility of developing novel rabies vaccines based on the self-replicating PRPs.FundingThis work was supported by the National Key Research and Development Program of China (2016YFD0500400).

Biodegradable PLGA-b-PEG Nanoparticles Induce T Helper 2 (Th2) Immune Responses and Sustained Antibody Titers via TLR9 Stimulation.

Sustained immune responses, particularly antibody responses, are key for protection against many endemic infectious diseases. Antibody responses are often accompanied by T helper (Th) cell immunity. Herein we study small biodegradable poly (ethylene glycol)-b-poly (lactic-co-glycolic acid) nanoparticles (PEG-b-PLGA NPs, 25–50 nm) as antigen- or adjuvant-carriers. The antigen carrier function of PEG-b-PLGA NPs was compared against an experimental benchmark polystyrene nanoparticles (PS NPs, 40–50 nm), both conjugated with the model antigen ovalbumin (OVA-PS NPs, and OVA-PEG-b-PLGA NPs). The OVA-PEG-b-PLGA NPs induced sustained antibody responses to Day 120 after two immunizations. The OVA-PEG-b-PLGA NPs as a self-adjuvanting vaccine further induced IL-4 producing T-helper cells (Th2), but not IFN-γ producing T-cells (Th1). The PEG-b-PLGA NPs as a carrier for CpG adjuvant (CpG-PEG-b-PLGA NPs) were also tested as mix-in vaccine adjuvants comparatively for protein antigens, or for protein-conjugated to PS NPs or to PEG-b-PLGA NPs. While the addition of this adjuvant NP did not further increase T-cell responses, it improved the consistency of antibody responses across all immunization groups. Together these data support further development of PEG-b-PLGA NPs as a vaccine carrier, particularly where it is desired to induce Th2 immunity and achieve sustained antibody titers in the absence of affecting Th1 immunity.

SAT-661 The Lesser Evil: Pembrolizumab Induced Remission of Metastatic Melanoma with Autoimmune Diabetes, Hypothyroidism, and Colitis

Background: Immune checkpoint inhibitors (ICI) are a new class of immunotherapy agents that empower the innate immune system to destroy cancer cells by overcoming checkpoints against autoimmunity and self-tolerance. ICI use against certain cancers has yielded impressive results. Pembrolizumab, an ICI approved by the FDA in 2014 for treatment of metastatic melanoma, is a monoclonal antibody which binds the programmed cell death protein 1 (PD1) receptor, blocking it’s activation. Recent case reports have demonstrated a link between pembrolizumab and severe immunotherapy related adverse events including the incidence of autoimmune disorders. Clinical Case: A 68-year-old male with hypertension and a past medical history of cutaneous melanoma treated fifteen years earlier with tumor resection was started on pembrolizumab after identification of new metastases to the lung and bones. After 3 cycles of therapy he began to experience dizziness, lightheadedness, and vision changes with diarrhea. Outpatient labs demonstrated hyperkalemia with hyponatremia. He was directed to the emergency department by his oncologist for presumed adrenal insufficiency, where he received one dose of IV dexamethasone (4 mg). Repeat labs demonstrated severe diabetic ketoacidosis with elevated blood glucose 981 (70-99 mg/dL), pH 7.19 (7.32-7.42), anion gap of 37 (4-15 mmol/L), and betahydroxybutyrate > 9.00 (0.00-0.40 mmol/L). Following endocrinology consultation steroids were discontinued in favor of iv insulin and fluid resuscitation, with admission to the medical intensive care unit. Further lab testing demonstrated low C-peptide levels and positive IA-2 and GAD-65 antibodies, confirming autoimmune diabetes. Endoscopic biopsy was also consistent with autoimmune colitis, and TSH one month after discharge was 123.70 (0.30-5.00 mcIU/mL) with free T4 < 0.1 (0.6-1.6 ng/dL).Despite early discontinuation of anti-PD1 therapy the melanoma has remained in remission for three years, suggesting a sustained immune response. He continues to require insulin and thyroid hormone replacement, though the autoimmune colitis has resolved. Conclusion: This case demonstrates the overall benefit of immune checkpoint inhibitor therapy in the treatment of metastatic melanoma, while highlighting a potentially lethal therapy complication with the concurrent onset of multiple autoimmune processes affecting separate organ systems. Increased awareness of the potential for DKA in patients not previously diagnosed with diabetes is needed to avoid delays in care and improve outcomes. This case also suggests a potential benefit to integrating routine blood glucose monitoring in immune checkpoint inhibitor treatment protocols, the utility of which should be further investigated.

Subchronic inhalation of Aspergillus versicolor conidia leads to sustained innate immune responses and is associated with irreversible remodeling of pulmonary arterial tissue following recovery

Abstract Aspergillus versicolor is an environmentally ubiquitous fungal species that is particularly abundant in environments with water infiltration. Respiration of fungal products, including conidia, hyphal fragments, mycotoxins, and other secondary metabolites may cause adverse pulmonary health effects including asthma and allergy. In the present study, pulmonary immune responses in B6C3F1 mice were examined following 1, 2, 4, 8, and 13 weeks of repeated respiratory challenge to 3×105A. versicolor conidia. Increased alveolar macrophages, inflammatory monocytes, neutrophils, and eosinophils, which peaked between 4 and 8 weeks, were detected in the lungs of mice exposed to viable conidia. Type 2 innate lymphoid cells (ILC2s) peaked between 8 and 13 weeks compared to total T cells which increased between 2 and 8 weeks and decreased at 13 weeks. Protein concentrations of the Th2 cytokines IL4, IL5, and IL13 were highest in bronchoaveolar lavage fluid of exposed mice following 4 weeks and exhibited a time-dependent reduction at 8 and 13 weeks. Inhalation of conidia for 13 weeks led to goblet cell hyperplasia, vascular wall thickening, and perivascular inflammation. Finally, mixed perivascular inflammation and smooth muscle hypertrophy were sustained at 4 weeks post-exposure. These data indicate that exposure to A. versicolor elicits a Th2-biased immune response that is driven by innate cell populations and is associated with the induction of remodeling of the pulmonary vasculature.

Conventional type 1 dendritic cells and natural killer cells demonstrate strong correlation to cytotoxic T lymphocyte infiltration in cervical cancer tumors

Abstract Professional antigen presenting cells such as dendritic cells (DCs) are vital for priming naive CD8+ T cells and developing a sustainable anti-tumor immune response. Natural killer (NK) cells within the tumor microenvironment (TME) recruit a specific population of DCs called conventional type 1 DCs (cDC1s). However, these cells are low in abundance making their detection in the tissue context challenging. To interrogate the presence of cDC1 and NK cells in the TME and reveal their spatial relationship to each other we utilized the highly sensitive and specific RNAscope Multiplex Fluorescence in situ hybridization (ISH) assay. NK cells and cDC1 cells were identified by using cell specific marker probes in 4 cervical cancer samples. Similarly, CTLs were visualized to determine if there is a correlation between the presence of cDC1 and NK cells and infiltration of CTLs within the cervical cancer tumors. Our results revealed a strong correlation between the presence of NK cells, cDC1 cells, and CTLs within 3 out of 4 cervical cancer samples. The NK cells showed expression of the chemokines XCL1 and CCL5, suggesting that the XCR1+/CCR5+ cDC1 cells may have been potentially recruited by these NK cells in the TME. Regions high in cDC1 and NK cells also showed significantly higher levels of CTL recruitment, indicated by the presence of CD8+/IFNG+ T cells. Conversely, 1 of the 4 cervical cancer samples demonstrated relatively lower levels of NK cells which correlated with lower cDC1 cells and in turn lower CTL infiltration. In conclusion, by utilizing the RNAscope Multiplex ISH assay we identified and visualized the spatial relationship between NK cells, CTLs, and cDC1 cells, highlighting the strength of this technology to spatially interrogate the TME.

Understanding how immune maturity shapes responses to cancer therapy

Abstract Clinical translation of cancer therapies from mice to human remains a constant challenge. Cancer immunotherapy aims develop a sustained long-term adaptive immune response to tumors. This raises the question of whether the typical pathogen-free animal models reflect the human immunological context and cancer pathology. Recent research suggests that exposure of pathogen-free mice to pet shop mice leads to their immunological maturation. We hypothesize that such a model may be more reflective of a human adult context. Specific-pathogen-free (SPF) Balb/c mice were co-housed with pet shop mice for 40 days to generate the “dirty mouse” model. Dirty mice were challenged with Listeria m. infection to test immune maturity. In parallel, CT26LacZ, CT26WT, EMT6 and K7M2 tumor cell lines were used to measure tumor growth in dirty mice. In particular, CT26LacZ tumor model was treated with oncolytic virus to assess the impact of immune maturity over efficacy of therapy. Complementary comparative experiments were performed in SPF mice. Dirty mouse immune response to Listeria challenge was more robust than in clean mice. Immune phenotyping revealed a different distribution of immune populations. A matured immune system impacted the growth of tumors to varying extents, with some models experiencing complete tumor rejection. Finally, oncolytic virus in dirty mice implanted with CT26-LacZ showed an improved efficacy on survival and tumor burden. Immune response to infections and tumor models between SPF mice and dirty mice reveals the potential impact of a mature immune system on immune-therapeutic responses. Therefore, establishment of model that better recreates the state of immune maturity may result in higher chances of translational impact.

A Sustained Immune Response Supports Long-Term Antiviral Immune Priming in the Pacific Oyster, Crassostrea gigas.

Over the last decade, innate immune priming has been evidenced in many invertebrate phyla. If mechanistic models have been proposed, molecular studies aiming to substantiate these models have remained scarce. We reveal here the transcriptional signature associated with immune priming in the oyster Crassostrea gigas Oysters were fully protected against Ostreid herpesvirus 1 (OsHV-1), a major oyster pathogen, after priming with poly(I·C), which mimics viral double-stranded RNA. Global analysis through RNA sequencing of oyster and viral genes after immune priming and viral infection revealed that poly(I·C) induces a strong antiviral response that impairs OsHV-1 replication. Protection is based on a sustained upregulation of immune genes, notably genes involved in the interferon pathway and apoptosis, which control subsequent viral infection. This persistent antiviral alert state remains active over 4 months and supports antiviral protection in the long term. This acquired resistance mechanism reinforces the molecular foundations of the sustained response model of immune priming. It further opens the way to applications (pseudovaccination) to cope with a recurrent disease that causes dramatic economic losses in the shellfish farming industry worldwide.IMPORTANCE In the last decade, important discoveries have shown that resistance to reinfection can be achieved without a functional adaptive immune system, introducing the concept of innate immune memory in invertebrates. However, this field has been constrained by the limited number of molecular mechanisms evidenced to support these phenomena. Taking advantage of an invertebrate species, the Pacific oyster (Crassostrea gigas), in which we evidenced one of the longest and most effective periods of protection against viral infection observed in an invertebrate, we provide the first comprehensive transcriptomic analysis of antiviral innate immune priming. We show that priming with poly(I·C) induced a massive upregulation of immune-related genes, which control subsequent viral infection, and it was maintained for over 4 months after priming. This acquired resistant mechanism reinforces the molecular foundations of the sustained response model of immune priming. It opens the way to pseudovaccination to prevent the recurrent diseases that currently afflict economically or ecologically important invertebrates.

Clinical and serological evaluation of capybaras (Hydrochoerus hydrochaeris) successively exposed to an Amblyomma sculptum-derived strain of Rickettsia rickettsii

Brazilian spotted fever (BSF), caused by Rickettsia rickettsii, is the most lethal tick-borne disease in the western hemisphere. In Brazil, Amblyomma sculptum ticks are the main vector. Capybaras (Hydrochoerus hydrochaeris), the largest living rodents of the world (adults weighing up to 100 Kg), have been recognized as amplifying hosts of R. rickettsii for A. sculptum in BSF-endemic areas; i.e., once primarily infected, capybaras develop bacteremia for a few days, when feeding ticks acquire rickettsial infection. We conducted experimental infections of five capybaras with an A. sculptum-derived strain of R. rickettsii and performed clinical and bacteremia evaluation during primary and subsequent infections. Bacteremia was detected in all capybaras during primary infection, but not in subsequent infections. All animals seroconverted to R. rickettsii (titres range: 64–32,768), and remained seropositive throughout the study. Primary infection resulted in clinical spotted fever illness in four capybaras, of which two had a fatal outcome. Subsequent infections in seropositive capybaras resulted in no clinical signs. Capybaras developed a sustained immune response that prevented a second bacteremia. This condition may imply a high reproduction rate of capybaras in BSF-endemic areas, in order to continuously generate capybaras susceptible to bacteremia during primary infection.

Immunopathology in the brain of mice following vertical transmission of Coxsackievirus B4

Coxsackie B viruses (CV–B) are associated with several central nervous system (CNS) disorders. These viruses are predominantly transmitted by fecal-oral route but vertical transmission can also occur. This work attempted to study the immune response ensuing vertical transmission of CV-B to the brain, and its eventual implementation in the brain pathogenesis. To this end, pregnant Swiss albino mice were inoculated with CV-B4 E2 or with sterile medium for control animals. At different ages after birth, brains were collected and analyzed for virus infection, histopathological changes and immune response. Infectious particles were detected in offspring's brain which demonstrates vertical transmission of the virus. This infection is persistent since the long lasting detection of viral RNA in offspring's brain. Some pathological signs including meningitis, edema and accumulation of inflammatory cells within and surrounding the inflammatory areas were observed. Immunoflorescence staining unveiled the presence of T lymphocytes and microgliosis in the sites of lesion for a long period after birth. Multiplex cytokines measurement upon supernatants of in vitro mixed brain cells and extracted mononuclear cells from offspring's brain has demonstrated an elevated secretion of the pro-inflammatory cytokines TNFα, IL-6 and IFNα and the chemokines RANTES and MCP-1. Hence, vertical transmission of CV-B4 and its persistence within offspring's brain can lead to pathological features linked to increased and sustained immune response.

Current laboratory diagnostics of coronavirus disease 2019 (COVID-19).

Laboratory medicine provides an almost irreplaceable contribution to the diagnostic reasoning and managed care of most human pathologies. The novel coronavirus disease 2019 (COVID-19) is not an exception to this paradigm. Although the relatively recent emergence does not allow to draw definitive conclusions on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostics, some standpoints can be conveyed. First and foremost, it seems now clear that we will be living together with this virus for quite a long time, so that our vigilance and responsiveness against the emergence of new local outbreaks shall be maintained at the highest possible levels. The etiological diagnosis of COVID-19 is, and will remain for the foreseeable future, deeply based on direct identification of viral RNA by means of molecular biology techniques in biological materials, especially upper and lower respiratory tract specimens. Whether other materials, such as blood, urine, stools, saliva and throat washing, will become valid alternatives has not been unequivocally defined so far. As concerns serological testing, promising information can be garnered from preliminary investigations, showing that the vast majority of COVID-19 patients seem to develop a sustained immune response against the virus, characterized especially by emergence of anti-SARS-CoV-2 IgG and IgA, 1 to 2 weeks after the onset of fever and/or respiratory symptoms. Whether these antibodies will have persistent neutralizing activity against the virus is still to be elucidated on individual and general basis. The availability of rapid tests for detecting either viral antigens or anti-SARS-CoV-2 antibodies are a potentially viable opportunity for purposes of epidemiologic surveillance, though more information is needed on accuracy and reliability of these portable immunoassays. (www.actabiomedica.it)

Protocols for Innate Lymphoid Cell Phenotypic and Functional Characterization: An Overview.

The innate immune system forms a first line of defense against infections and is an essential component in immune defense prior to the generation of sustained adaptive immune responses. Until recently this innate response was believed to be predominantly elicited by cells of myeloid origin. The last 10years have seen the discovery of an extensive family of innate lymphoid cells (ILCs) characterized by the ability to express cytokines, traditionally associated with activated T effector cells, and lack of expression of antigen receptors.ILCs are enriched at barrier surfaces and rapidly respond to alarmins within the tissue microenvironment. ILCs lack recombinant activating gene (RAG)-dependent rearranged receptors, lack myeloid and dendritic cell lineage defining factors, and possess lymphoid morphology. The first characterized innate lymphoid cells were natural killer cells (NK) cells and lymphoid tissue inducer (LTi) cells. NK cells, defined in 1975), mediate important early antigen independent immune responses. By contrast, LTi cells, defined in 1997, are vital for formation of lymph nodes during embryogenesis. In the past decade, research on ILCs has defined them as important regulators of barrier immunity. However, ILC phenotype and functional characterization is complex and requires highly specific protocols for delineating ILC specific function from other well-characterized immune cells. Within this book, each chapter provides an in-depth protocol that will expand on techniques used by laboratories to study ILC development, characterization and function in mice and humans.

Development of a Human Cytomegalovirus (HCMV)-Based Therapeutic Cancer Vaccine Uncovers a Previously Unsuspected Viral Block of MHC Class I Antigen Presentation.

Human cytomegalovirus (HCMV) induces a uniquely high frequency of virus-specific effector/memory CD8+ T-cells, a phenomenon termed “memory inflation”. Thus, HCMV-based vaccines are particularly interesting in order to stimulate a sustained and strong cellular immune response against cancer. Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with high lethality and inevitable relapse. The current standard treatment does not significantly improve the desperate situation underlining the urgent need to develop novel approaches. Although HCMV is highly fastidious with regard to species and cell type, GBM cell lines are susceptible to HCMV. In order to generate HCMV-based therapeutic vaccine candidates, we deleted all HCMV-encoded proteins (immunoevasins) that interfere with MHC class I presentation. The aim being to use the viral vector as an adjuvant for presentation of endogenous tumor antigens, the presentation of high levels of vector-encoded neoantigens and finally the repurposing of bystander HCMV-specific CD8+ T cells to fight the tumor. As neoantigen, we exemplarily used the E6 and E7 proteins of human papillomavirus type 16 (HPV-16) as a non-transforming fusion protein (E6/E7) that covers all relevant antigenic peptides. Surprisingly, GBM cells infected with E6/E7-expressing HCMV-vectors failed to stimulate E6-specific T cells despite high level expression of E6/E7 protein. Further experiments revealed that MHC class I presentation of E6/E7 is impaired by the HCMV-vector although it lacks all known immunoevasins. We also generated HCMV-based vectors that express E6-derived peptide fused to HCMV proteins. GBM cells infected with these vectors efficiently stimulated E6-specific T cells. Thus, fusion of antigenic sequences to HCMV proteins is required for efficient presentation via MHC class I molecules during infection. Taken together, these results provide the preclinical basis for development of HCMV-based vaccines and also reveal a novel HCMV-encoded block of MHC class I presentation.

Sustained Cellular and Humoral Immune Responses From an Adenoviral Vector-based Respiratory Syncytial Virus Vaccine.

Sustained Cellular and Humoral Immune Responses From an Adenoviral Vector-based Respiratory Syncytial Virus Vaccine.