Receptor transfer between immune cells by autoantibody-enhanced, CD32-driven trogocytosis is hijacked by HIV-1 to infect resting CD4 T cells.

Immune cell phenotyping frequently detects lineage-unrelated receptors. Here, we report that surface receptors can be transferred from primary macrophages to CD4 T cells and identify the Fcγ receptor CD32 as driver and cargo of this trogocytotic transfer. Filamentous CD32+ nanoprotrusions deposit distinct plasma membrane patches onto target T cells. Transferred receptors confer cell migration and adhesion properties, and macrophage-derived membrane patches render resting CD4 T cells susceptible to infection by serving as hotspots for HIV-1 binding. Antibodies that recognize T cell epitopes enhance CD32-mediated trogocytosis. Such autoreactive anti-HIV-1 envelope antibodies can be found in the blood of HIV-1 patients and, consistently, the percentage of CD32+ CD4 T cells is increased in their blood. This CD32-mediated, antigen-independent cell communication mode transiently expands the receptor repertoire and functionality of immune cells. HIV-1 hijacks this mechanism by triggering the generation of trogocytosis-promoting autoantibodies to gain access to immune cells critical to its persistence.

IFNα Subtypes in HIV Infection and Immunity.

Type I interferons (IFN), immediately triggered following most viral infections, play a pivotal role in direct antiviral immunity and act as a bridge between innate and adaptive immune responses. However, numerous viruses have evolved evasion strategies against IFN responses, prompting the exploration of therapeutic alternatives for viral infections. Within the type I IFN family, 12 IFNα subtypes exist, all binding to the same receptor but displaying significant variations in their biological activities. Currently, clinical treatments for chronic virus infections predominantly rely on a single IFNα subtype (IFNα2a/b). However, the efficacy of this therapeutic treatment is relatively limited, particularly in the context of Human Immunodeficiency Virus (HIV) infection. Recent investigations have delved into alternative IFNα subtypes, identifying certain subtypes as highly potent, and their antiviral and immunomodulatory properties have been extensively characterized. This review consolidates recent findings on the roles of individual IFNα subtypes during HIV and Simian Immunodeficiency Virus (SIV) infections. It encompasses their induction in the context of HIV/SIV infection, their antiretroviral activity, and the diverse regulation of the immune response against HIV by distinct IFNα subtypes. These insights may pave the way for innovative strategies in HIV cure or functional cure studies.

Influenza virus infection enhances tumour-specific CD8+ T-cell immunity, facilitating tumour control.

Influenza A virus (IAV) can cause severe respiratory infection leading to significant global morbidity and mortality through seasonal epidemics. Likewise, the constantly increasing number of cancer diseases is a growing problem. Nevertheless, the understanding of the mutual interactions of the immune responses between cancer and infection is still very vague. Therefore, it is important to understand the immunological cross talk between cancer and IAV infection. In several preclinical mouse models of cancer, including melanoma and colorectal cancer, we observed that IAV infection in the lung significantly decreased the tumour burden. Concomitantly, tumour-specific CD8+ T-cells are strongly activated upon infection, both in the tumour tissue and in the lung. CD8+ T-cell depletion during infection reverses the reduced tumour growth. Interestingly, IAV infection orchestrated the migration of tumour-specific CD8+ T-cells from the tumour into the infected lung. Blocking the migration of CD8+ T-cells prevented the anti-tumoural effect. Thus, our findings show that viral respiratory infection has significant impact on the anti-tumour CD8+ T-cell response, which will significantly improve our understanding of the immunological cross talk between cancer and infection.

Tg1.4HBV-s-rec mice, a crossbred hepatitis B virus-transgenic model, develop mild hepatitis.

Hepatitis B virus (HBV)-transgenic mice exhibit competent innate immunity and are therefore an ideal model for considering intrinsic or cell-based mechanisms in HBV pathophysiology. A highly replicative model that has been little used, let alone characterized, is the Tg1.4HBV-s-rec strain derived from cross breeding of HBV-transgenic mouse models that either accumulate (Alb/HBs, Tg[Alb1-HBV]Bri44) or lack (Tg1.4HBV-s-mut) the hepatitis B surface antigen (HBsAg). Tg1.4HBV-s-rec hepatocytes secreted HBsAg, Hepatitis B extracellular antigen (HBeAg) and produced HBV virions. Transmission electron microscopy visualised viral particles (Tg1.4HBV-s-rec), nuclear capsid formations (Tg1.4HBV-s-mut and Tg1.4HBV-s-rec) and endoplasmic reticulum malformations (Alb/HBs). Viral replication in Tg1.4HBV-s-rec and Tg1.4HBV-s-mut differed in HBsAg expression and interestingly in the distribution of HBV core antigen (HBcAg) and HBV × protein. While in Tg1.4HBV-s-mut hepatocytes, the HBcAg was located in the cytoplasm, in Tg1.4HBV-s-rec hepatocytes, the HBcAg appeared in the nuclei, suggesting a more productive replication. Finally, Tg1.4HBV-s-rec mice showed symptoms of mild hepatitis, with reduced liver function and elevated serum transaminases, which appeared to be related to natural killer T cell activation. In conclusion, the study of Alb/HBs, Tg1.4HBV-s-mut and their F1 progeny provides a powerful tool to elucidate HBV pathophysiology, especially in the early HBeAg-positive phases of chronic infection and chronic hepatitis.

Macrophage migration inhibitory factor receptor CD74 expression is associated with expansion and differentiation of effector T cells in COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused millions of COVID-19 cases and deaths worldwide. Severity of pulmonary pathologies and poor prognosis were reported to be associated with the activation non-virus-specific bystander T cells. In addition, high concentrations of the macrophage migration inhibitory factor (MIF) were found in serum of COVID-19 patients. We hypothesized that these two pathogenic factors might be related and analyzed the expression of receptors for MIF on T cells in COVID-19. T cells from PBMCs of hospitalized patients with mild and severe COVID-19 were characterized. A significantly higher proportion of CD4+ and CD8+ T cells from COVID-19 patients expressed CD74 on the cell surface compared to healthy controls. To induce intracellular signaling upon MIF binding, CD74 forms complexes with CD44, CXCR2, or CXCR4. The vast majority of CD74+ T cells expressed CD44, whereas expression of CXCR2 and CXCR4 was low in controls but increased upon SARS-CoV-2 infection. Hence, T cells in COVID-19 patients express receptors that render them responsive to MIF. A detailed analysis of CD74+ T cell populations revealed that most of them had a central memory phenotype early in infection, while cells with an effector and effector memory phenotype arose later during infection. Furthermore, CD74+ T cells produced more cytotoxic molecules and proliferation markers. Our data provide new insights into the MIF receptor and co-receptor repertoire of bystander T cells in COVID-19 and uncovers a novel and potentially druggable aspect of the immunological footprint of SARS-CoV-2.

Targeted mutations in IFNα2 improve its antiviral activity against various viruses.

During viral infections, type I interferons (IFN) are induced and play a key role in counteracting initial viral spread. Twelve different human IFNα subtypes exist that bind the same receptor; however, they elicit unique host responses and display distinct potencies of antiviral activities. Our previous studies on human immunodeficiency virus (HIV) and hepatitis B virus (HBV) demonstrated that the clinically used IFNα2 is not the most effective one among the IFNα subtypes. By sequence modeling, we identified a region in helix B with mainly conserved residues at the outside facing IFNAR1, but variable residues at the inside facing the core of IFNα, potentially representing a putative tunable anchor to tune pleiotropic IFN responses. Using site-directed mutagenesis, various mutations were introduced into the IFNα2b backbone targeting sites which are important for binding to IFNAR1 and IFNAR2, the putative tunable anchor, or outside these three regions. Selected mutations were based on sequence differences to high antiviral subtypes IFNα6 and IFNα14. Treatment assays against HBV and HIV identified several critical residues for the antiviral activity of IFNα mainly in the IFNAR1 binding region. Combined mutations of the IFNα2 IFNAR1/2 binding sites or the IFNAR1 binding region plus the putative tunable anchor by those of IFNα14 further augmented activation of different downstream signaling cascades providing a molecular correlate for the enhanced antiviral activity. We describe here important functional residues within IFNα subtype molecules, which enabled us to design novel and innovative drugs that may have the potential to be used in clinical trials against a variety of different viral infections.IMPORTANCEThe potency of interferon (IFN)α to restrict viruses was already discovered in 1957. However, until today, only IFNα2 out of the 12 distinct human IFNα subtypes has been therapeutically used against chronic viral infections. There is convincing evidence that other IFNα subtypes are far more efficient than IFNα2 against many viruses. In order to identify critical antiviral residues within the IFNα subtype sequence, we designed hybrid molecules based on the IFNα2 backbone with individual sequence motifs from the more potent subtypes IFNα6 and IFNα14. In different antiviral assays with HIV or HBV, residues binding to IFNAR1 as well as combinations of residues in the IFNAR1 binding region, the putative tunable anchor, and residues outside these regions were identified to be crucial for the antiviral activity of IFNα. Thus, we designed artificial IFNα molecules, based on the clinically approved IFNα2 backbone, but with highly improved antiviral activity against several viruses.

Immunotherapy-induced cytotoxic T follicular helper cells reduce numbers of retrovirus-infected reservoir cells in B cell follicles.

Antiretroviral therapy (ART) transformed HIV from a life-threatening disease to a chronic condition. However, eliminating the virus remains an elusive therapy goal. For several decades, Friend virus (FV) infection serves as a murine model to study retrovirus immunity. Similar to HIV, FV persists at low levels in lymph nodes B cell follicles avoiding elimination by immune cells. Such immune-privileged reservoirs exclude cytotoxic T cells from entry. However, CXCR5+ T cells are permitted to traffic through germinal centers. This marker is predominantly expressed by CD4+ follicular helper T cells (Tfh). Therefore, we explored immunotherapy to induce cytotoxic Tfh, which are rarely found under physiological conditions. The TNF receptor family member CD137 was first identified as a promising target for cancer immunotherapy. We demonstrated that FV-infected mice treatment with αCD137 antibody resulted in an induction of the cytotoxic program in Tfh. The therapy significantly increased numbers of cytotoxic Tfh within B cell follicles and contributed to viral load reduction. Moreover, αCD137 antibody combined with ART delayed virus rebound upon treatment termination without disturbing the lymph node architecture or antibody responses. Thus, αCD137 antibody therapy might be a novel strategy to target the retroviral reservoir and an interesting approach for HIV cure research.

SARS-CoV-2-specific T cell responses wane profoundly in convalescent individuals 10 months after primary infection.

A key question in the coronavirus disease 2019 (COVID-19) pandemic is the duration of specific T cell responses against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) post primary infection, which is difficult to address due to the large-scale COVID-19 vaccination and re-exposure to the virus. Here, we conducted an analysis of the long-term SARS-CoV-2-specific T cell responses in a unique cohort of convalescent individuals (CIs) that were among the first to be infected worldwide and without any possible antigen re-exposure since then. The magnitude and breadth of SARS-CoV-2-specific T cell responses correlated inversely with the time that had elapsed from disease onset and the age of those CIs. The mean magnitude of SARS-CoV-2-specific CD4 and CD8 T cell responses decreased about 82% and 76%, respectively, over the time period of ten months after infection. Accordingly, the longitudinal analysis also demonstrated that SARS-CoV-2-specific T cell responses waned significantly in 75% of CIs during the follow-up. Collectively, we provide a comprehensive characterization of the long-term memory T cell response in CIs, suggesting that robust SARS-CoV-2-specific T cell immunity post primary infection may be less durable than previously expected.

A systematic comparison reveals dynamic differences in early adaptive immune responses of acute-resolving versus chronic HBV replication.

Chronic hepatitis B virus (HBV) infection has been characterized by lack of effective adaptive immune responses which are vital for the viral clearance. However, very little is known about the dynamics of adaptive immune responses during the early phase of chronic HBV infection especially in spleen and liver. Here, we used the hydrodynamic injection (HDI) mouse model to kinetically characterize differences in the features of adaptive immunity, including the frequencies, phenotypes and function of antigen-presenting cells and T cells in the spleen, peripheral blood mononuclear cells (PBMCs) and liver, of chronic versus acute-resolving HBV replication (AR). We found that mice with AR mice and mice with chronic HBV replication (CH) mice showed early splenomegaly accompanied by T cell expansion in spleen but not in liver after HDI. Interestingly, the early and continuous increase in HBV-specific CD8+ T cells in spleen of CH mice was comparable to that in the AR mice. However, the splenic T cells of CH mice showed no activation phenotype compared with those in AR mice. Besides, increases in activated effector CD8+ T cells in PBMCs and liver at later time points were only observed in AR mice but not CH mice. CH mice also showed insufficient expansion of dendritic cells (DCs) in spleen and increased programmed death-1 expression in DCs of the liver compared to AR mice. The adoptive transfer of total splenocytes or splenic CD8+ T cells of AR mice to CH mice demonstrated that their ability to break HBV tolerance varies at different stages of HBV clearance. Moreover, the adoptive transfer of splenocytes from AR mice induce functional activation of endogenous HBV-specific CD8+ T cells of CH mice. Our results suggest that early T cell priming and expansion initially happens in the periphery after HBV antigen exposure in acute-resolving and chronic replication. The paucity of T cell activation, and subsequent migration and liver infiltration is a key feature of the adaptive immune responses during the early phase of CH, which is probably caused by the dysfunction of DCs.

SRSF1 acts as an IFN-I-regulated cellular dependency factor decisively affecting HIV-1 post-integration steps.

Efficient HIV-1 replication depends on balanced levels of host cell components including cellular splicing factors as the family of serine/arginine-rich splicing factors (SRSF, 1-10). Type I interferons (IFN-I) play a crucial role in the innate immunity against HIV-1 by inducing the expression of IFN-stimulated genes (ISGs) including potent host restriction factors. The less well known IFN-repressed genes (IRepGs) might additionally affect viral replication by downregulating host dependency factors that are essential for the viral life cycle; however, so far, the knowledge about IRepGs involved in HIV-1 infection is very limited. In this work, we could demonstrate that HIV-1 infection and the associated ISG induction correlated with low SRSF1 levels in intestinal lamina propria mononuclear cells (LPMCs) and peripheral blood mononuclear cells (PBMCs) during acute and chronic HIV-1 infection. In HIV-1-susceptible cell lines as well as primary monocyte-derived macrophages (MDMs), expression levels of SRSF1 were transiently repressed upon treatment with specific IFNα subtypes in vitro. Mechanically, 4sU labeling of newly transcribed mRNAs revealed that IFN-mediated SRSF1 repression is regulated on early RNA level. SRSF1 knockdown led to an increase in total viral RNA levels, but the relative proportion of the HIV-1 viral infectivity factor (Vif) coding transcripts, which is essential to counteract APOBEC3G-mediated host restriction, was significantly reduced. In the presence of high APOBEC3G levels, however, increased LTR activity upon SRSF1 knockdown facilitated the overall replication, despite decreased vif mRNA levels. In contrast, SRSF1 overexpression significantly impaired HIV-1 post-integration steps including LTR transcription, alternative splice site usage, and virus particle production. Since balanced SRSF1 levels are crucial for efficient viral replication, our data highlight the so far undescribed role of SRSF1 acting as an IFN-modulated cellular dependency factor decisively regulating HIV-1 post-integration steps.

CD47 restricts antiviral function of alveolar macrophages during influenza virus infection.

CD47 is an ubiquitously expressed surface molecule with significant impact on immune responses. However, its role for antiviral immunity is not fully understood. Here, we revealed that the expression of CD47 on immune cells seemed to disturb the antiviral immune response as CD47-deficient mice (CD47-/-) showed an augmented clearance of influenza A virus (IAV). Specifically, we have shown that enhanced viral clearance is mediated by alveolar macrophages (aMФ). Although aMФ displayed upregulation of CD47 expression during IAV infection in wildtype mice, depletion of aMФ in CD47-/- mice during IAV infection reversed the augmented viral clearance. We have also demonstrated that CD47 restricts hemoglobin (HB) expression in aMФ after IAV and severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection, with HB showing antiviral properties by enhancing the IFN-ß response. Our study showed a negative role for CD47 during antiviral immune responses in the lung by confining HB expression in aMФ.

Superior antiviral activity of IFNß in genital HSV-1 infection.

Type I interferons (IFNs) present the first line of defense against viral infections, providing antiviral, immunomodulatory and antiproliferative effects. The type I IFN family contains 12 IFNα subtypes and IFNß, and although they share the same receptor, they are classified as non-redundant, capable to induce a variety of different IFN-stimulated genes. However, the biological impact of individual subtypes remains controversial. Recent data propose a subtype-specificity of type I IFNs revealing unique effector functions for different viruses and thus expanding the implications for IFNα-based antiviral immunotherapies. Despite extensive research, drug-resistant infections with herpes simplex virus type 1 (HSV-1), which is the common agent of recurrent orogenital lesions, are still lacking a protective or curing therapeutic. However, due to the risk of generalized infections in immunocompromised hosts as well as the increasing incidence of resistance to conventional antiherpetic agents, HSV infections raise major health concerns. Based on their pleiotropic effector functions, the application of type I IFNs represents a promising approach to inhibit HSV-1 replication, to improve host immunity and to further elucidate their qualitative differences. Here, selective IFNα subtypes and IFNß were evaluated for their therapeutic potential in genital HSV-1 infections. Respective in vivo studies in mice revealed subtype-specific differences in the reduction of local viral loads. IFNß had the strongest antiviral efficacy against genital HSV-1 infection in mice, whereas IFNα1, IFNα4, and IFNα11 had no impact on viral loads. Based on flow cytometric analyses of underlying immune responses at local and peripheral sites, these differences could be further assigned to specific modulations of the antiviral immunity early during HSV-1 infection. IFNß led to enhanced systemic cytokine secretion and elevated cytotoxic responses, which negatively correlated with viral loads in the vaginal tract. These data provide further insights into the diversity of type I IFN effector functions and their impact on the immunological control of HSV-1 infections.

IFNα subtype-specific susceptibility of HBV in the course of chronic infection.

Chronic hepatitis B virus (HBV) infection continues to be a major health problem worldwide and remains hard to be cured. Therapy with interferon (IFN) α is an important method for the clinical treatment of chronic hepatitis B. IFNα exhibits direct antiviral effects as well as immunomodulatory activities, which can induce sustained antiviral responses in part of the treated chronic hepatitis B patients. Numerous IFNα subtypes with high sequence identity between 76-96% exist which are characterized by diverse, non-redundant biological activities. Our previous studies have demonstrated that the clinically approved IFNα2 is not the most effective subtype for the anti-HBV treatment among all IFNα subtypes. So far very little is known about the IFNα subtype expression pattern during early HBV infection and the IFNα subtype-specific susceptibility during persistent HBV infection as well as its related cellular mechanism. Here we determined the Ifna subtype mRNA expression during acute and chronic HBV infection by using the well-established hydrodynamic injection (HDI) mouse model and we revealed a transient but strong expression of a panel of Ifna subtypes in the spleen of HBV persistent replication mice compared to HDI controls. Immunotherapy with distinct IFNα subtypes controlled chronic HBV infection. IFNα subtype-mediated antiviral response and immune activation were comprehensively analyzed in an AAV-HBV persistent infection murine model and murine IFNα2 was identified as the most effective subtype in suppression of HBV replication. Further analysis of the immune response revealed a strong immunomodulatory activity of murine IFNα2 on splenic and intrahepatic NK and T cell activation during persistent HBV infection. Taken together, our data provide IFNα subtype-specific differences in the antiviral and immunomodulatory effector responses and a strong expression of all IFNα subtypes in the spleen during persistent HBV infection in mice. This knowledge will support the development of novel immunotherapeutic strategies for chronic hepatitis B infection.

The Bovine Seminal Plasma Protein PDC-109 Possesses Pan-Antiviral Activity.

Mammalian seminal plasma contains a multitude of bioactive components, including lipids, glucose, mineral elements, metabolites, proteins, cytokines, and growth factors, with various functions during insemination and fertilization. The seminal plasma protein PDC-109 is one of the major soluble components of the bovine ejaculate and is crucially important for sperm motility, capacitation, and acrosome reaction. A hitherto underappreciated function of seminal plasma is its anti-microbial and antiviral activity, which may limit the sexual transmission of infectious diseases during intercourse. We have recently discovered that PDC-109 inhibits the membrane fusion activity of influenza virus particles and significantly impairs viral infections at micromolar concentrations. Here we investigated whether the antiviral activity of PDC-109 is restricted to Influenza or if other mammalian viruses are similarly affected. We focused on Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), the etiological agent of the Coronavirus Disease 19 (COVID-19), thoroughly assessing PDC-109 inhibition with SARS-CoV-2 Spike (S)-pseudotyped reporter virus particles, but also live-virus infections. Consistent with our previous publications, we found significant virus inhibition, albeit accompanied by substantial cytotoxicity. However, using time-of-addition experiments we discovered a treatment regimen that enables virus suppression without affecting cell viability. We furthermore demonstrated that PDC-109 is also able to impair infections mediated by the VSV glycoprotein (VSVg), thus indicating a broad pan-antiviral activity against multiple virus species and families.

A ribavirin-induced ORF2 single-nucleotide variant produces defective hepatitis E virus particles with immune decoy function.

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and is the leading cause of enterically transmitted viral hepatitis worldwide. Ribavirin (RBV) is currently the only treatment option for many patients; however, cases of treatment failures or posttreatment relapses have been frequently reported. RBV therapy was shown to be associated with an increase in HEV genome heterogeneity and the emergence of distinct HEV variants. In this study, we analyzed the impact of eight patient-derived open reading frame 2 (ORF2) single-nucleotide variants (SNVs), which occurred under RBV treatment, on the replication cycle and pathogenesis of HEV. The parental HEV strain and seven ORF2 variants showed comparable levels of RNA replication in human hepatoma cells and primary human hepatocytes. However, a P79S ORF2 variant demonstrated reduced RNA copy numbers released in the supernatant and an impairment in the production of infectious particles. Biophysical and biochemical characterization revealed that this SNV caused defective, smaller HEV particles with a loss of infectiousness. Furthermore, the P79S variant displayed an altered subcellular distribution of the ORF2 protein and was able to interfere with antibody-mediated neutralization of HEV in a competition assay. In conclusion, an SNV in the HEV ORF2 could be identified that resulted in altered virus particles that were noninfectious in vitro and in vivo, but could potentially serve as immune decoys. These findings provide insights in understanding the biology of circulating HEV variants and may guide development of personalized antiviral strategies in the future.

Distinct Type I Interferon Subtypes Differentially Stimulate T Cell Responses in HIV-1-Infected Individuals.

The expression of type I interferons (IFNs) is one of the immediate host responses during most viral infections. The type I IFN family consists of numerous highly conserved IFNα subtypes, IFNß, and some others. Although these IFNα subtypes were initially believed to act interchangeably, their discrete biological properties are nowadays widely accepted. Subtype-specific antiviral, immunomodulatory, and anti-proliferative activities were reported explained by differences in receptor affinity, downstream signaling events, and individual IFN-stimulated gene expression patterns. Type I IFNs and increased IFN signatures potentially linked to hyperimmune activation of T cells are critically discussed for chronic HIV (human immunodeficiency virus) infection. Here, we aimed to analyze the broad immunological effects of specific type I IFN subtypes (IFNα2, IFNα14, and IFNß) on T and NK cell subsets during HIV-1 infection in vitro and ex vivo. Stimulation with IFNα14 and IFNß significantly increased frequencies of degranulating (CD107a+) gut-derived CD4+ T cells and blood-derived T and NK cells. However, frequencies of IFNγ-expressing T cells were strongly reduced after stimulation with IFNα14 and IFNß. Phosphorylation of downstream molecules was not only IFN subtype-specific; also, significant differences in STAT5 phosphorylation were observed in both healthy peripheral blood mononuclear cells (PBMCs) and PBMCs of HIV-infected individuals, but this effect was less pronounced in healthy gut-derived lamina propria mononuclear cells (LPMCs), assuming cell and tissue specific discrepancies. In conclusion, we observed distinct type I IFN subtype-specific potencies in stimulating T and NK cell responses during HIV-1-infection.

IFNAR1 Deficiency Impairs Immunostimulatory Properties of Neutrophils in Tumor-Draining Lymph Nodes.

Tumor-draining lymph nodes (TDLNs) are the first organs where the metastatic spread of different types of cancer, including head and neck cancer (HNC), occurs and have therefore high prognostic relevance. Moreover, first anti-cancer immune responses have been shown to be initiated in such LNs via tumor-educated myeloid cells. Among myeloid cells present in TDLNs, neutrophils represent a valuable population and considerably participate in the activation of effector lymphocytes there. Tumor-supportive or tumor-inhibiting activity of neutrophils strongly depends on the surrounding microenvironment. Thus, type I interferon (IFN) availability has been shown to prime anti-tumor activity of these cells. In accordance, mice deficient in type I IFNs show elevated tumor growth and metastatic spread, accompanied by the pro-tumoral neutrophil bias. To reveal the mechanism responsible for this phenomenon, we have studied here the influence of defective type I IFN signaling on the immunoregulatory activity of neutrophils in TDLNs. Live imaging of such LNs was performed using two-photon microscopy in a transplantable murine HNC model. CatchupIVM-red and Ifnar1-/- (type I IFN receptor- deficient) CatchupIVM-red mice were used to visualize neutrophils and to assess their interaction with T-cells in vivo. We have evaluated spatiotemporal patterns of neutrophil/T-cell interactions in LNs in the context of type I interferon receptor (IFNAR1) availability in tumor-free and tumor-bearing animals. Moreover, phenotypic and functional analyses were performed to further characterize the mechanisms regulating neutrophil immunoregulatory capacity. We demonstrated that inactive IFNAR1 leads to elevated accumulation of neutrophils in TDLNs. However, these neutrophils show significantly impaired capacity to interact with and to stimulate T-cells. As a result, a significant reduction of contacts between neutrophils and T lymphocytes is observed, with further impairment of T-cell proliferation and activation. This possibly contributes to the enhanced tumor growth in Ifnar1-/- mice. In agreement with this, IFNAR1-independent activation of downstream IFN signaling using IFN-λ improved the immunostimulatory capacity of neutrophils in TDLNs and contributed to the suppression of tumor growth. Our results suggest that functional type I IFN signaling is essential for neutrophil immunostimulatory capacity and that stimulation of this signaling may provide a therapeutic opportunity in head and neck cancer patients.

Differential interferon-α subtype induced immune signatures are associated with suppression of SARS-CoV-2 infection.

Type I interferons (IFN-I) exert pleiotropic biological effects during viral infections, balancing virus control versus immune-mediated pathologies, and have been successfully employed for the treatment of viral diseases. Humans express 12 IFN-alpha (α) subtypes, which activate downstream signaling cascades and result in distinct patterns of immune responses and differential antiviral responses. Inborn errors in IFN-I immunity and the presence of anti-IFN autoantibodies account for very severe courses of COVID-19; therefore, early administration of IFN-I may be protective against life-threatening disease. Here we comprehensively analyzed the antiviral activity of all IFNα subtypes against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to identify the underlying immune signatures and explore their therapeutic potential. Prophylaxis of primary human airway epithelial cells (hAEC) with different IFNα subtypes during SARS-CoV-2 infection uncovered distinct functional classes with high, intermediate, and low antiviral IFNs. In particular, IFNα5 showed superior antiviral activity against SARS-CoV-2 infection in vitro and in SARS-CoV-2-infected mice in vivo. Dose dependency studies further displayed additive effects upon coadministration with the broad antiviral drug remdesivir in cell culture. Transcriptomic analysis of IFN-treated hAEC revealed different transcriptional signatures, uncovering distinct, intersecting, and prototypical genes of individual IFNα subtypes. Global proteomic analyses systematically assessed the abundance of specific antiviral key effector molecules which are involved in IFN-I signaling pathways, negative regulation of viral processes, and immune effector processes for the potent antiviral IFNα5. Taken together, our data provide a systemic, multimodular definition of antiviral host responses mediated by defined IFN-I. This knowledge will support the development of novel therapeutic approaches against SARS-CoV-2.

Distinct effects of treatment with two different interferon-alpha subtypes on HIV-1-associated T-cell activation and dysfunction in humanized mice.

OBJECTIVE: Interferon-alpha (IFN-α) has been associated with excessive immune activation and dysfunction during HIV-1 infection. However, evidence suggests specific IFN-α subtypes may be beneficial rather than detrimental. This study compared the effects of treatment with two different IFN-α subtypes on indicators of T-cell activation and dysfunction during HIV-1 infection. DESIGN: Humanized mice were infected with HIV-1 for 5 weeks and then treated with two different IFN-α subtypes for an additional 3 weeks. Splenic T cells were assessed both immediately posttreatment and again 6 weeks after treatment cessation. METHODS: HIV-1 infected triple-knockout bone marrow-liver-thymus mice received daily intraperitoneal injections of either IFN-α14 or the clinically approved subtype, IFN-α2. T cells were analysed directly ex vivo for indicators of activation and dysfunction or stimulated to determine their proliferative capacity and ability to produce functional mediators. RESULTS: Unlike IFN-α2, IFN-α14 treatment reduced viremia and resulted in less activated CD4+ T cells and a lower naïve to effector CD8+ T-cell ratio. Despite exhibiting a reduced proliferative response, the frequency of CD8+ T cells from IFN-α14 treated mice that produced functional mediators and expressed markers of dysfunction was more similar to healthy controls than untreated and IFN-α2 treated mice. Frequencies of exhaustion marker expression remained higher in untreated and IFN-α2 treated mice 6 weeks posttreatment despite similar viral loads between groups at this timepoint. CONCLUSIONS: Treatment with different IFN-α subtypes had distinctive effects on T cells during HIV-1 infection. IFN-α14 was associated with fewer indicators of T-cell dysfunction whereas IFN-α2 treatment had little impact.

Longitudinal characterization of phenotypic profile of T cells in chronic hepatitis B identifies immune markers associated with HBsAg loss.

BACKGROUND: The current desirable endpoint of treatment against chronic hepatitis B virus infection (cHBV) is to achieve a functional cure, which is defined as HBsAg loss (sAg-L) with or without anti-HBs seroconversion. However, the immunological features that are associated with functional cure have not been studied in detail. METHODS: 172 cHBV patients (67 HBeAg+ and 105 HBeAg-), including 141 HBsAg retained (sAg-R) patients (115 chronic hepatitis and 26 asymptomatic carriers), 31 sAg-L patients, and 24 healthy individuals (vaccinated but not infected with HBV) were examined for their T cell phenotypic profile and HBV-specific T cell responses by flow cytometry. 18 cHBV patients with low serum HBsAg levels were also longitudinally followed for their T cell phenotypic profile and HBV-specific T cell responses up to 60 weeks. FINDINGS: sAg-L patients showed distinct CD4+ and CD8+ T cell phenotype fingerprints compared to those of sAg-R patients, as mainly indicated by the upregulation of HLA-DR on both CD4+ and CD8+ T cells, and a potent HBcAg-specific CD8+ T cell response. The changes in the T cell phenotype in cHBV patients were even more profound during rapid HBsAg decrease and was associated with interferon α treatment. The expression of HLA-DR (r = 0·3269, p = 0·0037), CD95 (r = 0·2796, p = 0·0151), CD40L (r = 0·2747, p = 0·0156), CTLA-4 (r = 0·2786, p = 0·0148), TIM-3 (r = 0·3082, p = 0·0068), CD107a (r = 0·3597, p = 0·0013) on CD4+ T cells, and HLA-DR (r = 0·3542, p = 0·0016), CD69 (r = 0·2507, p = 0·0279), CD107a (r = 0·2875, p = 0·0112) on CD8+ T cells were positively correlated with the rate of HBsAg decrease. The expression of HLA-DR (r = 0·2846, p = 0·0009) and CD95 (r = 0·2442, p = 0·0049) on CD8+ T cells were positively correlated with the magnitude of the HBcAg-specific T cell responses in cHBV patients. Importantly, CTLA-4, CD95 and CD107a expression on CD4+ T cells, as well as HLA-DR and TIM-3 expression on CD8+ T cells in combination with HBsAg quantification were identified as potential predictive factors for sAg-L within 48 weeks in cHBV patients. INTERPRETATION: The onset of HBsAg decrease and subsequent loss in cHBV patients on treatment is associated with significant alterations of both CD4+ and CD8+ T cell phenotypes. Characterization of the T cell phenotype in cHBV patients may present predicative value for sAg-L. FUNDING: National Natural Science Foundation of China, National Scientific and Technological Major Project of China, Integrated Innovative Team for Major Human Diseases Program of Tongji Medical College, "Double-First Class" Project for the International Cooperation Center on Infection and Immunity, HUST.