IKK2/NFkB signaling controls lung resident CD8+ T cell memory during influenza infection.

CD8+ T cell tissue resident memory (TRM) cells are especially suited to control pathogen spread at mucosal sites. However, their maintenance in lung is short-lived. TCR-dependent NFkB signaling is crucial for T cell memory but how and when NFkB signaling modulates tissue resident and circulating T cell memory during the immune response is unknown. Here, we find that enhancing NFkB signaling in T cells once memory to influenza is established, increases pro-survival Bcl-2 and CD122 levels thus boosting lung CD8+ TRM maintenance. By contrast, enhancing NFkB signals during the contraction phase of the response leads to a defect in CD8+ TRM differentiation without impairing recirculating memory subsets. Specifically, inducible activation of NFkB via constitutive active IKK2 or TNF interferes with TGFß signaling, resulting in defects of lung CD8+ TRM imprinting molecules CD69, CD103, Runx3 and Eomes. Conversely, inhibiting NFkB signals not only recovers but improves the transcriptional signature and generation of lung CD8+ TRM. Thus, NFkB signaling is a critical regulator of tissue resident memory, whose levels can be tuned at specific times during infection to boost lung CD8+ TRM.

STING controls T cell memory fitness during infection through T cell-intrinsic and IDO-dependent mechanisms.

Stimulator of interferon genes (STING) signaling has been extensively studied in inflammatory diseases and cancer, while its role in T cell responses to infection is unclear. Using Listeria monocytogenes strains engineered to induce different levels of c-di-AMP, we found that high STING signals impaired T cell memory upon infection via increased Bim levels and apoptosis. Unexpectedly, reduction of TCR signal strength or T cell-STING expression decreased Bim expression, T cell apoptosis, and recovered T cell memory. We found that TCR signal intensity coupled STING signal strength to the unfolded protein response (UPR) and T cell survival. Under strong STING signaling, Indoleamine-pyrrole 2,3-dioxygenase (IDO) inhibition also reduced apoptosis and led to a recovery of T cell memory in STING sufficient CD8 T cells. Thus, STING signaling regulates CD8 T cell memory fitness through both cell-intrinsic and extrinsic mechanisms. These studies provide insight into how IDO and STING therapies could improve long-term T cell protective immunity.

Interplay of Inflammatory, Antigen and Tissue-Derived Signals in the Development of Resident CD8 Memory T Cells.

CD8 positive, tissue resident memory T cells (TRM) are a specialized subset of CD8 memory T cells that surveil tissues and provide critical first-line protection against tumors and pathogen re-infection. Recently, much effort has been dedicated to understanding the function, phenotype and development of TRM. A myriad of signals is involved in the development and maintenance of resident memory T cells in tissue. Much of the initial research focused on the roles tissue-derived signals play in the development of TRM, including TGFß and IL-33 which are critical for the upregulation of CD69 and CD103. However, more recent data suggest further roles for antigenic and pro-inflammatory cytokines. This review will focus on the interplay of pro-inflammatory, tissue and antigenic signals in the establishment of resident memory T cells.

Sphingosine kinase 2 restricts T cell immunopathology but permits viral persistence.

Chronic viral infections are often established by the exploitation of immune-regulatory mechanisms that result in nonfunctional T cell responses. Viruses that establish persistent infections remain a serious threat to human health. Sphingosine kinase 2 (SphK2) generates sphingosine 1-phosphate, which is a molecule known to regulate multiple cellular processes. However, little is known about SphK2's role during the host immune responses to viral infection. Here, we demonstrate that SphK2 functions during lymphocytic choriomeningitis virus Cl 13 (LCMV Cl 13) infection to limit T cell immune pathology, which subsequently aids in the establishment of virus-induced immunosuppression and the resultant viral persistence. The infection of Sphk2-deficient (Sphk2-/-) mice with LCMV Cl 13 led to the development of nephropathy and mortality via T cell-mediated immunopathology. Following LCMV infection, Sphk2-/- CD4+ T cells displayed increased activity and proliferation, and these cells promoted overactive LCMV Cl 13-specific CD8+ T cell responses. Notably, oral instillation of an SphK2-selective inhibitor promoted protective T cell responses and accelerated the termination of LCMV Cl 13 persistence in mice. Thus, SphK2 is indicated as an immunotherapeutic target for the control of persistent viral infections.

NFκB-Pim-1-Eomesodermin axis is critical for maintaining CD8 T-cell memory quality.

T-cell memory is critical for long-term immunity. However, the factors involved in maintaining the persistence, function, and phenotype of the memory pool are undefined. Eomesodermin (Eomes) is required for the establishment of the memory pool. Here, we show that in T cells transitioning to memory, the expression of high levels of Eomes is not constitutive but rather requires a continuum of cell-intrinsic NFκB signaling. Failure to maintain NFκB signals after the peak of the response led to impaired Eomes expression and a defect in the maintenance of CD8 T-cell memory. Strikingly, we found that antigen receptor [T-cell receptor (TCR)] signaling regulates this process through expression of the NFκB-dependent kinase proviral integration site for Moloney murine leukemia virus-1 (PIM-1), which in turn regulates NFκB and Eomes. T cells defective in TCR-dependent NFκB signaling were impaired in late expression of Pim-1, Eomes, and CD8 memory. These defects were rescued when TCR-dependent NFκB signaling was restored. We also found that NFκB-Pim-1 signals were required at memory to maintain memory CD8 T-cell longevity, effector function, and Eomes expression. Hence, an NFκB-Pim-1-Eomes axis regulates Eomes levels to maintain memory fitness.

Hemagglutinin of Influenza A Virus Antagonizes Type I Interferon (IFN) Responses by Inducing Degradation of Type I IFN Receptor 1.

UNLABELLED: Influenza A virus (IAV) employs diverse strategies to circumvent type I interferon (IFN) responses, particularly by inhibiting the synthesis of type I IFNs. However, it is poorly understood if and how IAV regulates the type I IFN receptor (IFNAR)-mediated signaling mode. In this study, we demonstrate that IAV induces the degradation of IFNAR subunit 1 (IFNAR1) to attenuate the type I IFN-induced antiviral signaling pathway. Following infection, the level of IFNAR1 protein, but not mRNA, decreased. Indeed, IFNAR1 was phosphorylated and ubiquitinated by IAV infection, which resulted in IFNAR1 elimination. The transiently overexpressed IFNAR1 displayed antiviral activity by inhibiting virus replication. Importantly, the hemagglutinin (HA) protein of IAV was proved to trigger the ubiquitination of IFNAR1, diminishing the levels of IFNAR1. Further, influenza A viral HA1 subunit, but not HA2 subunit, downregulated IFNAR1. However, viral HA-mediated degradation of IFNAR1 was not caused by the endoplasmic reticulum (ER) stress response. IAV HA robustly reduced cellular sensitivity to type I IFNs, suppressing the activation of STAT1/STAT2 and induction of IFN-stimulated antiviral proteins. Taken together, our findings suggest that IAV HA causes IFNAR1 degradation, which in turn helps the virus escape the powerful innate immune system. Thus, the research elucidated an influenza viral mechanism for eluding the IFNAR signaling pathway, which could provide new insights into the interplay between influenza virus and host innate immunity. IMPORTANCE: Influenza A virus (IAV) infection causes significant morbidity and mortality worldwide and remains a major health concern. When triggered by influenza viral infection, host cells produce type I interferon (IFN) to block viral replication. Although IAV was shown to have diverse strategies to evade this powerful, IFN-mediated antiviral response, it is not well-defined if IAV manipulates the IFN receptor-mediated signaling pathway. Here, we uncovered that influenza viral hemagglutinin (HA) protein causes the degradation of type I IFN receptor subunit 1 (IFNAR1). HA promoted phosphorylation and polyubiquitination of IFNAR1, which facilitated the degradation of this receptor. The HA-mediated elimination of IFNAR1 notably decreased the cells' sensitivities to type I IFNs, as demonstrated by the diminished expression of IFN-induced antiviral genes. This discovery could help us understand how IAV regulates the host innate immune response to create an environment optimized for viral survival in host cells.

Lactoferrin acts as an adjuvant during influenza vaccination of neonatal mice.

Health policy precludes neonatal vaccination against influenza. Hence, morbidity and mortality are high under 6 months of age. Lactoferrin may activate diminished numbers of dysfunctional dendritic cells and reverse neonatal vaccine failures. Aluminum hydroxide/ALUM recruits neutrophils that secrete lactoferrin at deposition sites of antigen. We theorized lactoferrin + influenza antigen initiates an equivalent antibody response compared to ALUM. Three-day-old mice received subcutaneously 30 µg of H1N1 hemagglutinin + 200 µg of bovine lactoferrin versus hemagglutinin + ALUM. Controls received hemagglutinin, lactoferrin, or ALUM. After 21 days, sera measured anti-H1N1 (ELISA) and neutralizing antibody (plaque assays). ELISA detected equal antibody production with lactoferrin + hemagglutinin compared to hemagglutinin + ALUM; both sera also neutralized H1N1 virus at a 1:20 dilution (p < 0.01). Controls had no anti-H1N1 antibody. Neonates given lactoferrin had no anaphylaxis when challenged four weeks later. Lactoferrin is a safe and effective adjuvant for inducing antibody against influenza in neonates.

A ceramide analogue stimulates dendritic cells to promote T cell responses upon virus infections.

The ceramide family of lipids plays important roles in both cell structure and signaling in a diverse array of cell types, including immune cells. However, very little is known regarding how ceramide affects the activation of dendritic cells (DCs) in response to viral infection. In this study, we demonstrate that a synthetic ceramide analog (C8) stimulates DCs to increase the expansion of virus-specific T cells upon virus infection. Exogenously supplied C8 ceramide elevated the expression of DC maturation markers such as MHC class I and costimulatory molecules following infection with the clone 13 strain of lymphocytic choriomeningitis virus (LCMV) or influenza virus. Importantly, ceramide-conditioned, LCMV-infected DCs displayed an increased ability to promote expansion of virus-specific CD8(+) T cells when compared with virus-infected DCs. Furthermore, a locally instilled ceramide analog significantly increased virus-reactive T cell responses in vivo to both LCMV and influenza virus infections. Collectively, these findings provide new insights into ceramide-mediated regulation of DC responses against virus infection and help us establish a foundation for novel immune-stimulatory therapeutics.

Sphingosine kinase 1 serves as a pro-viral factor by regulating viral RNA synthesis and nuclear export of viral ribonucleoprotein complex upon influenza virus infection.

Influenza continues to pose a threat to humans by causing significant morbidity and mortality. Thus, it is imperative to investigate mechanisms by which influenza virus manipulates the function of host factors and cellular signal pathways. In this study, we demonstrate that influenza virus increases the expression and activation of sphingosine kinase (SK) 1, which in turn regulates diverse cellular signaling pathways. Inhibition of SK suppressed virus-induced NF-κB activation and markedly reduced the synthesis of viral RNAs and proteins. Further, SK blockade interfered with activation of Ran-binding protein 3 (RanBP3), a cofactor of chromosome region maintenance 1 (CRM1), to inhibit CRM1-mediated nuclear export of the influenza viral ribonucleoprotein complex. In support of this observation, SK inhibition altered the phosphorylation of ERK, p90RSK, and AKT, which is the upstream signal of RanBP3/CRM1 activation. Collectively, these results indicate that SK is a key pro-viral factor regulating multiple cellular signal pathways triggered by influenza virus infection.

Sphingosine analogue AAL-R increases TLR7-mediated dendritic cell responses via p38 and type I IFN signaling pathways.

Sphingosine analogues display immunosuppressive activities and thus have therapeutic potential in the treatment of autoimmune diseases. In this study, we investigated the effects of the sphingosine analogue AAL-R (FTY720 derivative) on dendritic cell (DC) response upon TLR stimulation. Unlike its known immunosuppressive activity, AAL-R increased TLR7-mediated DC responses by elevating the levels of MHC class I and costimulatory molecules and type I IFN expression and by enhancing the capacity of DCs to induce CD8(+) T cell proliferation. Importantly, the stimulatory activity of AAL-R was dependent on type I IFN signaling, as type I IFN receptor-deficient DCs failed to respond to AAL-R. Also, AAL-R activated p38 MAPK to increase type I IFN synthesis and TLR7-mediated DC maturation. These findings enhance our understanding of sphingosine regulation of the host immune system, in particular upon pathogenic infections.