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Inhibiting PD-1:PD-L1 signaling has transformed therapeutic immune restoration. CD4+ T cells sustain immunity in chronic infections and cancer, yet little is known about how PD-1 signaling modulates CD4+ helper T (TH) cell responses or the ability to restore CD4+ TH-mediated immunity by checkpoint blockade. We demonstrate that PD-1:PD-L1 specifically suppressed CD4+ TH1 cell amplification, prevents CD4+ TH1 cytokine production and abolishes CD4+ cytotoxic killing capacity during chronic infection in mice. Inhibiting PD-L1 rapidly restored these functions, while simultaneously amplifying and activating TH1-like T regulatory cells, demonstrating a system-wide CD4–TH1 recalibration. This effect coincided with decreased T cell antigen receptor signaling, and re-directed type I interferon (IFN) signaling networks towards dominant IFN-γ-mediated responses. Mechanistically, PD-L1 blockade specifically targeted defined populations with pre-established, but actively suppressed proliferative potential, with limited impact on minimally cycling TCF-1+ follicular helper T cells, despite high PD-1 expression. Thus, CD4+ T cells require unique differentiation and functional states to be targets of PD-L1-directed suppression and therapeutic restoration. Snell et al. examine the heterogeneity of CD4+ T cells in chronic viral infection, showing that PD-L1 blockade enhances a cytotoxic gene program in antigen-specific TH1 cells and can restore antiviral CD4+ T cell killer function.

Background The accurate interpretation of RNA-Seq data presents a moving target as scientists continue to introduce new experimental techniques and analysis algorithms. Simulated datasets are an invaluable tool to accurately assess the performance of RNA-Seq analysis methods. However, existing RNA-Seq simulators focus on modeling the technical biases and artifacts of sequencing, rather than on simulating the original RNA samples. A first step in simulating RNA-Seq is to simulate RNA. Results To fill this need, we developed the Configurable And Modular Program Allowing RNA Expression Emulation (CAMPAREE), a simulator using empirical data to simulate diploid RNA samples at the level of individual molecules. We demonstrated CAMPAREE’s use for generating idealized coverage plots from real data, and for adding the ability to generate allele-specific data to existing RNA-Seq simulators that do not natively support this feature. Conclusions Separating input sample modeling from library preparation/sequencing offers added flexibility for both users and developers to mix-and-match different sample and sequencing simulators to suit their specific needs. Furthermore, the ability to maintain sample and sequencing simulators independently provides greater agility to incorporate new biological findings about transcriptomics and new developments in sequencing technologies. Additionally, by simulating at the level of individual molecules, CAMPAREE has the potential to model molecules transcribed from the same genes as a heterogeneous population of transcripts with different states of degradation and processing (splicing, editing, etc.). CAMPAREE was developed in Python, is open source, and freely available at https://github.com/itmat/CAMPAREE.

Serial circulating tumor DNA (ctDNA) monitoring is emerging as a non-invasive strategy to predict and monitor immune checkpoint blockade (ICB) therapeutic efficacy across cancer types. Yet, limited data exist to show the relationship between ctDNA dynamics and tumor genome and immune microenvironment in patients receiving ICB. Here, we present an in-depth analysis of clinical, whole-exome, transcriptome, and ctDNA profiles of 73 patients with advanced solid tumors, across 30 cancer types, from a phase II basket clinical trial of pembrolizumab (NCT02644369) and report changes in genomic and immune landscapes (primary outcomes). Patients stratified by ctDNA and tumor burden dynamics correspond with survival and clinical benefit. High mutation burden, high expression of immune signatures, and mutations in BRCA2 are associated with pembrolizumab molecular sensitivity, while abundant copy-number alterations and B2M loss-of-heterozygosity corresponded with resistance. Upon treatment, induction of genes expressed by T cell, B cell, and myeloid cell populations are consistent with sensitivity and resistance. We identified the upregulated expression of PLA2G2D, an immune-regulating phospholipase, as a potential biomarker of adaptive resistance to ICB. Together, these findings provide insights into the diversity of immunogenomic mechanisms that underpin pembrolizumab outcomes., Although circulating tumour DNA (ctDNA) can predict immune checkpoint blockade (ICB) responses, its association with tumour biomarkers remains unknown. Here, the authors use ctDNA to inform exome and transcriptome profiling of >100 patients with 30 cancer types on a single clinical ICB trial and identify tumour microenvironment features associated with response.

Micropolymorphisms within human leukocyte antigen (HLA) class I molecules can change the architecture of the peptide-binding cleft, leading to differences in peptide presentation and T cell recognition. The impact of such HLA variation on natural killer (NK) cell recognition remains unclear. Given the differential association of HLA-B*57:01 and HLA-B*57:03 with the control of HIV, recognition of these HLA-B57 allomorphs by the killer cell immunoglobulin-like receptor (KIR) 3DL1 was compared. Despite differing by only two polymorphic residues, both buried within the peptide-binding cleft, HLA-B*57:01 more potently inhibited NK cell activation. Direct-binding studies showed KIR3DL1 to preferentially recognize HLA-B*57:01, particularly when presenting peptides with positively charged position (P)Ω-2 residues. In HLA-B*57:01, charged PΩ-2 residues were oriented toward the peptide-binding cleft and away from KIR3DL1. In HLA-B*57:03, the charged PΩ-2 residues protruded out from the cleft and directly impacted KIR3DL1 engagement. Accordingly, KIR3DL1 recognition of HLA class I ligands is modulated by both the peptide sequence and conformation, as determined by the HLA polymorphic framework, providing a rationale for understanding differences in clinical associations.

Optimal lung repair and regeneration is essential for recovery from viral infections such as that induced by influenza A virus (IAV). We have previously demonstrated that lung inflammation induced by IAV is under circadian control. However, it is not known if the circadian clock exerts its influence on lung repair and regenerative processes independent of acute inflammation from IAV. Here, we demonstrate for the first time that lung organoids have a functional clock as they mature and that the absence of an intact circadian clock impairs regenerative capacity. Using several models of circadian disruption, we show that with the absence of an intact clock lung proliferation is disrupted. Further, we find that the circadian clock acts through direct control of the Wnt/β-catenin pathway. We speculate, that adding the circadian dimension to the critical process of lung repair and regeneration will lead to novel therapies and improve outcomes. Finally, we use data from UK Biobank to demonstrate at the population level, the role of poor circadian rhythms in mediating negative outcomes following lung infection.

Interferon-induced transmembrane (IFITM) proteins inhibit a broad range of enveloped viruses by blocking entry into host cells. We used an inducible overexpression system to investigate if IFITM1, IFITM2, and IFITM3 could modulate early and/or late stages of influenza A virus (IAV) or parainfluenza virus 3 (PIV-3) infection in human A549 airway epithelial cells. IAV and PIV-3 represent respiratory viruses which utilize distinct cellular entry pathways. We verify entry by endocytosis for IAV, whereas PIV-3 infection was consistent with fusion at the plasma membrane. Following induction prior to infection, all three IFITM proteins restricted the percentage of IAV-infected cells at 8 hours postinfection. In contrast, prior induction of IFITM1 and IFITM2 did not inhibit PIV-3 infection, although a modest reduction was observed with IFITM3. Small interfering RNA (siRNA)-mediated knockdown of endogenous IFITM1, IFITM2, and IFITM3 expression, in the presence or absence of pretreatment with type I interferon, resulted in increased IAV, but not PIV-3, infection. This finding suggests that while all three IFITMs display antiviral activity against IAV, they do not restrict the early stages of PIV-3 infection. IAV and PIV-3 infection culminates in viral egress through budding at the plasma membrane. Inducible expression of IFITM1, IFITM2, or IFITM3 immediately after infection did not impact titers of infectious virus released from IAV- or PIV-3-infected cells. Our findings show that IFITM proteins differentially restrict the early stages of infection of two respiratory viruses with distinct cellular entry pathways but do not influence the late stages of replication for either virus. IMPORTANCE Interferon-induced transmembrane (IFITM) proteins restrict the initial stages of infection for several respiratory viruses; however, their potential to modulate the later stages of virus replication has not been explored. In this study, we highlight the utility of an inducible overexpression system to assess the impact of IFITM proteins on either early- or late-stage replication of two respiratory viruses. We demonstrate antiviral activity by IFITM1, IFITM2, and IFITM3 against influenza A virus (IAV) but not parainfluenza virus 3 (PIV-3) during the early stages of cellular infection. Furthermore, IFITM induction following IAV or PIV-3 infection does not restrict the late stages of replication of either virus. Our findings show that IFITM proteins can differentially restrict the early stages of infection of two viruses with distinct cellular entry pathways and yet do not influence the late stages of replication for either virus.

Membrane-associated RING-CH8 (MARCH8) impairs the cell surface expression of envelope glycoproteins from different viruses, reducing their incorporation into virions. Using stable cell lines with inducible MARCH8 expression, we show that MARCH8 did not alter susceptibility to influenza A virus (IAV) infection, but virions released from infected cells were markedly less infectious. Knockdown of endogenous MARCH8 confirmed its effect on IAV infectivity. The expression of MARCH8 impaired the infectivity of both H3N2 and H1N1 strains and was dependent on its E3 ligase activity. Although virions released in the presence of MARCH8 expressed smaller amounts of viral hemagglutinin (HA) and neuraminidase (NA) proteins, there was no impact on levels of the viral HA, NA, or matrix 2 (M2) proteins detected on the surface of infected cells. Moreover, mutation of lysine residues in the cytoplasmic tails of HA, NA, and/or M2, or in the viral M1 protein, did not abrogate MARCH8-mediated restriction. While MARCH1 and -8 target similar immunological ligands and both restrict HIV-1, only MARCH8 inhibited IAV infectivity. Deletion of the N-terminal cytoplasmic (N-CT) domain of MARCH8 confirmed it to be a critical determinant of IAV inhibition. Of interest, deletion of the MARCH1 N-CT or its replacement with the MARCH8 N-CT resulted in acquisition of IAV restriction. Together, these data demonstrate that MARCH8 restricts a late stage in IAV replication by a mechanism distinct to its reported activity against other viruses. Moreover, we show that the N-CT of MARCH8 is essential for anti-IAV activity, whereas the MARCH1 N-CT inhibits its ability to restrict IAV. IMPORTANCE The antiviral activity of MARCH8 has been associated with the downregulation of envelope glycoproteins from a range of different viruses, resulting in reduced incorporation into nascent virions. Here, we show that MARCH8 restricts IAV at a late stage in virus replication, but this was not associated with reduced expression of IAV envelope glycoproteins on the surface of infected cells, pointing to a distinct mechanism of antiviral activity. Our studies also demonstrate the differential ability of MARCH1 and -8 to restrict IAV infectivity, highlighting the critical role of the N-CT domain of each protein in modulating IAV restriction. Overall, these studies provide novel insights regarding the mechanisms by which MARCH proteins contribute to cell-intrinsic immunity against IAV.

Abortive infection of macrophages serves as a "dead end" for most seasonal influenza A virus (IAV) strains, and it is likely to contribute to effective host defence. Interferon (IFN)-induced transmembrane protein 3 (IFITM3) restricts the early stages of IAV replication in epithelial cells, but IFITM3 restriction of IAV replication in macrophages has not been previously investigated. Herein, macrophages isolated from IFITM3-deficient mice were more susceptible to initial IAV infection, but late-stage viral replication was still controlled through abortive infection. Strikingly, IFNα/β receptor (IFNAR)-deficient macrophages infected with IAV were not only more susceptible to initial infection, but these cells also supported productive viral replication. Significantly, we have established that abortive IAV infection in macrophages is controlled through a type I IFN-dependent mechanism, where late-stage IAV replication can proceed in the absence of type I IFN responses. These findings provide novel mechanistic insight into macrophage-specific processes that potently shut down IAV replication.

Over 20 million Americans are affected by allergies such as asthma and allergic rhinitis, which can be caused by common allergens like house dust mites (HDM) or their associated components. The exacerbation of allergies can be fatal in patients with pre-existing chronic lung inflammation. Until now, there is no cure for asthma and type 2 allergies and the only available treatments aim to reduce the disease symptoms. Thus, there is an urgent need to understand the pathogenic mechanism of HDM-induced allergic responses. Since HDM play a major role in triggering allergic reactions, several studies focused on identifying their constituents. Although it is important to study HDM protein components that elicit adaptive type 2 inflammation (e.g. Der p 1), the role of innate sensors such as Toll-like receptors (TLRs) is unknown. A number of studies investigated presence of common adjuvants that may activate host immune receptors and modulate allergic diseases, for example, lipopolysaccharide (LPS) contaminating HDM triggered asthma in mice through acting on TLR4. In addition, single and double-stranded nucleic acids from bacteria or viruses can also act as adjuvants that stimulate innate immune receptors. Clearly, diverse allergen-associated components play an essential role in modulating type 2 lung inflammation in allergic patients. Recently, we have shown that HDM contain immunogenic RNA capable of modulating type 2 lung inflammation by inducing protective interferon responses. Using RNA-seq analysis, we show that HDM contain previously uncharacterized double stranded RNA sequences derived from novel RNA-viruses. Our findings suggest that other allergens may also have immunostimulatory RNA that can regulate type 2 lung inflammation.

Background In lung transplant recipients, immunosuppressive medications result in impaired antiviral immunity and a propensity for cytomegalovirus (CMV) reactivation within the lung allograft. Natural killer (NK) cells play a key role in immunity to CMV, with an increase in the proportion of NK cells expressing activating CD94-NKG2C receptors in the blood being a strong correlate of CMV infection. Whether a similar increase in NKG2C NK cells occurs in lung transplant recipients following CMV reactivation in the allograft and if such cells contribute to viral control remains unclear. Methods In this pilot study, we longitudinally assessed the frequency and phenotype of NKG2C NK cells in the blood and bronchoalveolar lavage (BAL) of lung transplant recipients and stratified recipients based on their risk of developing CMV disease. Results We observed an increase in the proportion of NKG2C NK cells in the blood and BAL of CMV high-risk patients, coincident with both the cessation of antiviral prophylaxis and subsequent detection of actively replicating CMV in the blood and lung allograft. Additionally, these NKG2C NK cells expressed killer-cell immunoglobulin-like receptors distinct from those of other NK subsets and BAL NKG2C NK cells possessed an activated phenotype. Finally, the frequency of NKG2C NK cells in the BAL may be inversely correlated with CMV blood titers. Conclusions Monitoring the phenotype of NK cells postlung transplant may be a useful biomarker for monitoring patient levels of CMV immunity.

Sezary syndrome (SS) is an aggressive cutaneous T cell lymphoma with pruritic skin inflammation and immune dysfunction, driven by neoplastic, clonal memory T cells in both peripheral blood and skin. To gain insight into abnormal gene expression promoting T cell dysfunction, lymphoproliferation and transformation in SS, we first compared functional transcriptomic profiles of both resting and activated CD4+CD45RO+ T cells from SS patients and normal donors to identified differential expressed genes. Next, a meta-analysis was performed to compare our SS data to public microarray data from a novel benign disease control, lymphocytic-variant hypereosinophilic syndrome (L-HES). L-HES is a rare, clonal lymphoproliferation of abnormal memory T cells that produces similar clinical symptoms as SS, including severe pruritus and eosinophilia. Comparison revealed gene sets specific for either SS (370 genes) or L-HES (519 genes), and a subset of 163 genes that were dysregulated in both SS and L-HES T cells compared to normal donor T cells. Genes confirmed by RT-qPCR included elevated expression of PLS3, TWIST1 and TOX only in SS, while IL17RB mRNA was increased only in L-HES. CDCA7 was increased in both diseases. In an L-HES patient who progressed to peripheral T cell lymphoma, the malignant transformation identified increases in the expression of CDCA7, TIGIT, and TOX, which are highly expressed in SS, suggesting that these genes contribute to neoplastic transformation. In summary, we have identified gene expression biomarkers that implicate a common transformative mechanism and others that are unique to differentiate SS from L-HES.

ObjectiveTo compare the early responses to joint injury in conventional and germ-free mice.DesignPost-traumatic osteoarthritis (PTOA) was induced using a non-invasive anterior cruciate ligament rupture model in 20-week old germ-free (GF) and conventional C57BL/6 mice. Injury was induced in the left knees of n=8 GF and n=10 conventional mice. To examine the effects of injury, n=5 GF and n=9 conventional naïve control mice were used. Mice were euthanized 7 days post-injury, followed by synovial fluid recovery for global metabolomic profiling and analysis of epiphyseal trabecular bone by micro-computed tomography (μCT). Global metabolomic profiling assessed metabolic differences in the joint response to injury between GF and conventional mice. Magnitude of trabecular bone volume loss measured using μCT assessed early OA progression in GF and conventional mice.ResultsμCT found that GF mice had significantly less trabecular bone loss compared to conventional mice, indicating that the GF status was protective against early OA changes in bone structure. Global metabolomic profiling showed that conventional mice had greater variability in their metabolic response to injury, and a more distinct joint metabolome compared to their corresponding controls. Furthermore, differences in the response to injury in GF compared to conventional mice were linked to mouse metabolic pathways that regulate inflammation associated with the innate immune system.ConclusionsThese results suggest that the gut microbiota promote the development of PTOA during the acute phase following joint trauma possibly through the regulation of the innate immune system.

During chronic viral infections, CD8 T cells rapidly lose antiviral and immune-stimulatory functions in a sustained program termed exhaustion. In addition to this loss of function, CD8 T cells with the highest affinity for viral antigen can be physically deleted. Consequently, treatments designed to restore function to exhausted cells and control chronic viral replication are limited from the onset by the decreased breadth of the antiviral T cell response. Yet, it remains unclear why certain populations of CD8 T cells are deleted while others are preserved in an exhausted state. We report that CD8 T cell deletion during chronic viral infection can be prevented by therapeutically lowering viral replication early after infection. The initial resistance to deletion enabled long-term maintenance of antiviral cytolytic activity of the otherwise deleted high-affinity CD8 T cells. In combination with decreased virus titers, CD4 T cell help and prolonged interactions with costimulatory molecules B7-1/B7-2 were required to prevent CD8 T cell deletion. Thus, therapeutic strategies to decrease early virus replication could enhance virus-specific CD8 T cell diversity and function during chronic infection.

Indigenous people worldwide are at high risk of developing severe influenza disease. HLA-A*24:02 allele, highly prevalent in Indigenous populations, is associated with influenza-induced mortality, although the basis for this association is unclear. Here, we define CD8+ T-cell immune landscapes against influenza A (IAV) and B (IBV) viruses in HLA-A*24:02-expressing Indigenous and non-Indigenous individuals, human tissues, influenza-infected patients and HLA-A*24:02-transgenic mice. We identify immunodominant protective CD8+ T-cell epitopes, one towards IAV and six towards IBV, with A24/PB2550–558-specific CD8+ T cells being cross-reactive between IAV and IBV. Memory CD8+ T cells towards these specificities are present in blood (CD27+CD45RA− phenotype) and tissues (CD103+CD69+ phenotype) of healthy individuals, and effector CD27−CD45RA−PD-1+CD38+CD8+ T cells in IAV/IBV patients. Our data show influenza-specific CD8+ T-cell responses in Indigenous Australians, and advocate for T-cell-mediated vaccines that target and boost the breadth of IAV/IBV-specific CD8+ T cells to protect high-risk HLA-A*24:02-expressing Indigenous and non-Indigenous populations from severe influenza disease.

We investigated the contribution of human leukocyte antigen A2 (HLA-A2) and HLA-E-restricted CD8+ T cells in patients with Mycobacterium tuberculosis and human immunodeficiency virus 1 (HIV-1) coinfection. HIV-1 downregulates HLA-A, -B, and -C molecules in infected cells, thus influencing recognition by HLA class I-restricted CD8+ T cells but not by HLA-E-restricted CD8+ T cells, owing to the inability of the virus to downmodulate their expression. Therefore, antigen-specific HLA-E-restricted CD8+ T cells could play a protective role in Mycobacterium tuberculosis and HIV-1 coinfection. HLA-E- and HLA-A2-restricted Mycobacterium tuberculosis-specific CD8+ T cells were tested in vitro for cytotoxic and microbicidal activities, and their frequencies and phenotypes were evaluated ex vivo in patients with active tuberculosis and concomitant HIV-1 infection. HIV-1 and Mycobacterium tuberculosis coinfection caused downmodulation of HLA-A2 expression in human monocyte-derived macrophages associated with resistance to lysis by HLA-A2-restricted CD8+ T cells and failure to restrict the growth of intracellular Mycobacterium tuberculosis. Conversely, HLA-E surface expression and HLA-E-restricted cytolytic and microbicidal CD8 responses were not affected. HLA-E-restricted and Mycobacterium tuberculosis-specific CD8+ T cells were expanded in the circulation of patients with Mycobacterium tuberculosis/HIV-1 coinfection, as measured by tetramer staining, but displayed a terminally differentiated and exhausted phenotype that was rescued in vitro by anti-PD-1 (programmed cell death protein 1) monoclonal antibody. Together, these results indicate that HLA-E-restricted and Mycobacterium tuberculosis-specific CD8+ T cells in patients with Mycobacterium tuberculosis/HIV-1 coinfection have an exhausted phenotype and fail to expand in vitro in response to antigen stimulation, which can be restored by blocking the PD-1 pathway using the specific monoclonal antibody nivolumab.

We describe a case of a young man, taking no other routine medications, presenting with erythema multiforme and cetirizine-induced psychosis with re-challenge evidence. On retrospective elicitation of history, it was found that he had been involved in a motor vehicle collision 4 months prior and was a daily cannabis user; there were no objective abnormalities by MRI and neurological evaluations. Although rare, cetirizine-induced psychosis is an important adverse drug reaction that warrants the attention of healthcare practitioners.

2'3'-cGAMP is known as a nonclassical second messenger and small immune modulator that possesses potent antitumor and antiviral activities via inducing the stimulator of IFN genes-mediated (STING-mediated) signaling pathway. However, its function in regulating type 2 immune responses remains unknown. Therefore, we sought to determine a role of STING activation by 2'3'-cGAMP in type 2 inflammatory reactions in multiple mouse models of eosinophilic asthma. We discovered that 2'3'-cGAMP administration strongly attenuated type 2 lung immunopathology and airway hyperreactivity induced by IL-33 and a fungal allergen, Aspergillus flavus. Mechanistically, upon the respiratory delivery, 2'3'-cGAMP was mainly internalized by alveolar macrophages, in which it activated the STING/IFN regulatory factor 3/type I IFN signaling axis to induce the production of inhibitory factors containing IFN-α, which blocked the IL-33-mediated activation of group 2 innate lymphoid (ILC2) cells in vivo. We further demonstrated that 2'3'-cGAMP directly suppressed the proliferation and function of both human and mouse ILC2 cells in vitro. Taken together, our findings suggest that STING activation by 2'3'-cGAMP in alveolar macrophages and ILC2 cells can negatively regulate type 2 immune responses, implying that the respiratory delivery of 2'3'-cGAMP might be further developed as an alternative strategy for treating type 2 immunopathologic diseases such as eosinophilic asthma.

Human cytomegalovirus (CMV) infection can stimulate robust human leukocyte antigen (HLA)-E-restricted CD8+ T cell responses. These T cells recognize a peptide from UL40, which differs by as little as a single methyl group from self-peptides that also bind HLA-E, challenging their capacity to avoid self-reactivity. Unexpectedly, we showed that the UL40/HLA-E T cell receptor (TCR) repertoire included TCRs that had high affinities for HLA-E/self-peptide. However, paradoxically, lower cytokine responses were observed from UL40/HLA-E T cells bearing TCRs with high affinity for HLA-E. RNA sequencing and flow cytometric analysis revealed that these T cells were marked by the expression of inhibitory natural killer cell receptors (NKRs) KIR2DL1 and KIR2DL2/L3. On the other hand, UL40/HLA-E T cells bearing lower-affinity TCRs expressed the activating receptor NKG2C. Activation of T cells bearing higher-affinity TCRs was regulated by the interaction between KIR2D receptors and HLA-C. These findings identify a role for NKR signaling in regulating self/non-self discrimination by HLA-E-restricted T cells, allowing for antiviral responses while avoiding contemporaneous self-reactivity.

Estradiol (E2) is a sex hormone which has been shown to be protective against sexually transmitted infections such as herpes simplex virus 2 (HSV-2). However, few studies have examined the underlying mechanisms by which this occurs. Here, we investigated the effect of E2 on the establishment of memory T cells post-intranasal immunization with HSV-2. CD4(+) T cell responses first appeared in the upper respiratory tract (URT) within 3 days postimmunization before being detected in the female reproductive tract (FRT) at 7 days. E2 treatment resulted in greater and earlier T(h)17 responses, which preceded augmented T(h)1 responses at these sites. The CD4(+) T cells persisted in the URT for up to 28 days, and E2 treatment resulted in higher frequencies of memory T cells. Intranasal immunization also led to the establishment of CD4(+) tissue-resident memory T cells (T(RM) cells) in the FRT, and E2 treatment resulted in increased T(h)1 and T(h)17 T(RM) cells. When the migration of circulating T cells into the FRT was blocked by FTY720, immunized E2-treated mice remained completely protected against subsequent genital HSV-2 challenge compared to non-E2 controls, confirming that T(RM) cells alone are adequate for protection in these mice. Finally, the enhanced vaginal T(h)1 T(RM) cells present in E2-treated mice were found to be modulated through an interleukin 17 (IL-17)-mediated pathway, as E2-treated IL-17A-deficient mice had impaired establishment of T(h)1 T(RM) cells. This study describes a novel role for E2 in enhancing CD4(+) memory T cells and provides insight on potential strategies for generating optimal immunity during vaccination. IMPORTANCE Herpes simplex virus 2 (HSV-2) is a highly prevalent sexually transmitted infection for which there is currently no vaccine available. Interestingly, the female sex hormone estradiol has been shown to be protective against HSV-2. However, the underlying mechanisms by which this occurs remains relatively unknown. Our study demonstrates that under the influence of estradiol treatment, intranasal immunization with an attenuated strain of HSV-2 leads to enhanced establishment of antiviral memory T cell responses in the upper respiratory tract and female reproductive tract. In these sites, estradiol treatment leads to greater T(h)17 memory cells, which precede enhanced T(h)1 memory responses. Consequently, the T cell responses mounted by tissue-resident memory cells in the female reproductive tract of estradiol-treated mice are sufficient to protect mice against vaginal HSV-2 challenge. This study offers important insights regarding the regulation of mucosal immunity by hormones and on potential strategies for generating optimal immunity during vaccination.

Indigenous people worldwide are at high-risk of developing severe influenza disease. HLA-A*24:02 allele, highly prevalent in Indigenous populations, is associated with influenza-induced mortality, although the basis for this association is unclear. We defined CD8+ T-cell immune landscapes against influenza A (IAV) and B (IBV) viruses in HLA-A*24:02-expressing Indigenous and non-Indigenous individuals, human tissues, influenza-infected patients and HLA-A*24:02-transgenic mice. We identified immunodominant protective CD8+ T-cell epitopes, one towards IAV and six towards IBV, with A24/PB2550-558-specific CD8+ T-cells cells being cross-reactive between IAV and IBV. Memory CD8+ T-cells towards these specificities were present in blood (CD27+CD45RA- phenotype) and tissues (CD103+CD69+ phenotype) of healthy subjects, and effector CD27-CD45RA-PD-1+CD38+CD8+ T-cells in IAV/IBV patients. Our data present the first evidence of influenza-specific CD8+ T-cell responses in Indigenous Australians, and advocate for T-cell-mediated vaccines that target and boost the breadth of IAV/IBV-specific CD8+ T-cells to protect high-risk HLA-A*24:02-expressing Indigenous and non-Indigenous populations from severe influenza disease.One Sentence SummaryInfluenza-specific CD8+ T-cell specificities restricted by HLA-A*24:02.

Mycoplasma pneumoniae is a major cause of community-acquired pneumonia. There are limited data in the United States on the molecular epidemiological characteristics of M. pneumoniae We collected 446 M. pneumoniae-positive specimens from 9 states between August 2012 and October 2018. Culture, antimicrobial susceptibility testing, P1 subtyping, and multilocus VNTR (variable-number tandem repeats) analysis (MLVA) were performed to characterize the isolates. Macrolide-resistant M. pneumoniae (MRMp) was detected in 37 (8.3%) specimens. P1 subtype 2 (P1-2) was the predominant P1 subtype (59.8%). P1 subtype distribution did not change significantly chronologically or geographically. The macrolide resistance rate in P1 subtype 1 (P1-1) samples was significantly higher than that in P1-2 (12.9% versus 5.5%). Six P1-2 variants were identified, including two novel types, and variant 2c was predominant (64.6%). P1-2 variants were distributed significantly differently among geographic regions. Classical P1-2 was more frequent in lower respiratory tract specimens and had longer p1 trinucleotide repeats. Classical P1-2 was most common in MRMp (35.7%), while variant 2c was most common in macrolide-susceptible M. pneumoniae (67.5%). Fifteen MLVA types were identified; 3-5-6-2 (41.7%), 4-5-7-2 (35.3%), and 3-6-6-2 (16.6%) were the major types, and four MLVA clusters were delineated. The distribution of MLVA types varied significantly over time and geographic location. The predominant MLVA type switched from 4-5-7-2 to 3-5-6-2 in 2015. MLVA type was associated with P1 subtypes and P1-2 variant types but not with macrolide resistance. To investigate the M. pneumoniae genotype shift and its impact on clinical presentations, additional surveillance programs targeting more diverse populations and prolonged sampling times are required.

BACKGROUND Anti-viral treatments to control cytomegalovirus (CMV) after lung transplantation (LTx) are associated with toxicity and anti-viral resistance. Cellular immunotherapy with virus-specific cytotoxic T cells has yielded promising results but requires donor/recipient matching. γδ T cells are involved in anti-viral immunity and can recognize antigens independently of major histocompatibility complex molecules and may not require the same level of matching. We assessed the phenotype of circulating γδ T cells after LTx to identify the candidate populations for CMV immunotherapy. METHODS Peripheral blood mononuclear cells were isolated from lung transplant recipients before transplantation and at routine bronchoscopies after LTx. Patients were stratified by risk of CMV disease into moderate risk (recipient CMV seropositive, n = 15) or high risk (HR) (recipient CMV seronegative/donor CMV seropositive, n = 10). CMV replication was classified as polymerase chain reaction positive (>150 copies/ml) in blood and/or bronchoalveolar lavage within the first 18 months. The phenotype of γδ T cells was assessed by multicolor flow cytometry, and T-cell receptor (TCR) sequences were determined by deep sequencing. RESULTS In HR lung transplant recipients with CMV replication, we observed striking phenotypic changes in γδ T cells, marked by an increase in the proportion of effector Vδ1+ γδ T cells expressing the activating natural killer cell receptor NKG2C. Moreover, we observed a remarkable increase in TCR diversity. CONCLUSIONS NKG2C+ Vδ1+ γδ T cells were associated with CMV replication and may indicate their potential to control infection. As such, we propose that they could be a potential target for cellular therapy against CMV.

Repeated exposures to environmental allergens in susceptible individuals drive the development of type 2 inflammatory conditions such as asthma, which have been traditionally considered to be mainly mediated by Th2 cells. However, emerging evidence suggest that a new innate cell type, group 2 innate lymphoid cells (ILC2), plays a central role in initiating and amplifying a type 2 response, even in the absence of adaptive immunity. At present, the regulatory mechanisms for controlling ILC2 activation remain poorly understood. Here we report that respiratory delivery of immunogenic extracellular RNA (exRNAs) derived from RNA- and DNA-virus infected cells, was able to activate a protective response against acute type 2 lung immunopathology and airway hyperresponsiveness (AHR) induced by IL-33 and a fungal allergen, A. flavus, in mice. Mechanistically, we found that the innate immune responses triggered by exRNAs had a potent suppressive effect in vivo on the proliferation and function of ILC2 without the involvement of adaptive immunity. We further provided the loss-of-function genetic evidence that the TLR3- and MAVS-mediated signaling axis is essential for the inhibitory effects of exRNAs in mouse lungs. Thus, our results indicate that the host detection of extracellular immunostimulatory RNAs generated during respiratory viral infections have an important function in the regulation of ILC2-driven acute lung inflammation.

International audience; We demonstrate that DNA hypomethylating agent (HMA) treatment can directly modulate the anti-tumor response and effector function of CD8(+) T cells. In vivo HMA treatment promotes CD8(+) T cell tumor infiltration and suppresses tumor growth via CD8(+) T cell-dependent activity. Ex vivo, HMAs enhance primary human CD8(+) T cell activation markers, effector cytokine production, and anti-tumor cytolytic activity. Epigenomic and transcriptomic profiling shows that HMAs vastly regulate T cell activation-related transcriptional networks, culminating with over-activation of NFATc1 short isoforms. Mechanistically, demethylation of an intragenic CpG island immediately downstream to the 3’ UTR of the short isoform was associated with antisense transcription and alternative polyadenylation of NFATc1 short isoforms. High-dimensional single-cell mass cytometry analyses reveal a selective effect of HMAs on a subset of human CD8(+) T cell subpopulations, increasing both the number and abundance of a granzyme B(high), perforin(high) effector subpopulation. Overall, our findings support the use of HMAs as a therapeutic strategy to boost anti-tumor immune response.

Ca2+ release–activated Ca2+ channel regulator 2A (CRACR2A) is expressed abundantly in T cells and acts as a signal transmitter between TCR stimulation and activation of the Ca2+/NFAT and JNK/AP1 pathways. CRACR2A has been linked to human diseases in numerous genome-wide association studies and was shown to be one of the most sensitive targets of the widely used statin drugs. However, the physiological role of CRACR2A in T cell functions remains unknown. In this study, using transgenic mice for tissue-specific deletion, we show that CRACR2A promotes Th1 responses and effector function of Th17 cells. CRACR2A was abundantly expressed in Th1 and Th17 cells. In vitro, deficiency of CRACR2A decreased Th1 differentiation under nonpolarizing conditions, whereas the presence of polarizing cytokines compensated this defect. Transcript analysis showed that weakened TCR signaling by deficiency of CRACR2A failed to promote Th1 transcriptional program. In vivo, conditional deletion of CRACR2A in T cells alleviated Th1 responses to acute lymphocytic choriomeningitis virus infection and imparted resistance to experimental autoimmune encephalomyelitis. Analysis of CNS from experimental autoimmune encephalomyelitis–induced mice showed impaired effector functions of both Th1 and Th17 cell types, which correlated with decreased pathogenicity. Collectively, our findings demonstrate the requirement of CRACR2A-mediated TCR signaling in Th1 responses as well as pathogenic conversion of Th17 cells, which occurs at the site of inflammation.

Eosinophilic esophagitis is an atopic disease defined clinically by esophageal symptoms in combination with a dense esophageal eosinophilia. EoE is triggered and maintained by exposure to certain foods and it is known that dietary modification controls symptoms and achieves disease remission. Recently, aeroallergens have been implicated in the pathogenesis of EoE. To examine the role of aeroallergens in EoE, we reviewed the published literature. Sensitization and production of IgE antibodies to foods and aeroallergens in subjects with EoE has been demonstrated. However, the evidence suggests only a minor role for IgE-mediated immune reactions in EoE. There is some evidence to support an association of EoE diagnosis and flares with environmental allergen exposure, and animal studies support the notion that EoE may be induced by exposure to inhalant allergens. Some studies show that newly diagnosed cases of EoE follow a seasonal pollen distribution (summer and spring), but the weight of evidence does not support the seasonal occurrence of diagnosis or worsening of symptoms. Overall, we conclude that the current evidence does not support causality in inhalant allergen exposure and the genesis nor exacerbations of EoE in humans, although there is a possibility that inhalant allergen sensitization could play a modifying role in EoE in the context of cross-reacting food allergens.

Human leukocyte antigen (HLA)-C*06:02 is identified as the allele associated with the highest risk for the development of the autoimmune skin disease psoriasis. However, the diversity and mode of peptide presentation by the HLA-C*06:02 molecule remains unclear. Here, we describe the endogenous peptide repertoire of ∼3,000 sequences for HLA-C*06:02 that defines the peptide-binding motif for this HLA allomorph. We found that HLA-C*06:02 predominantly presents nonamer peptides with dominant arginine anchors at the P2 and P7 positions and a preference for small hydrophobic residues at the C terminus (PΩ). To determine the structural basis of this selectivity, we determined crystal structures of HLA-C*06:02 in complex with two self-peptides (ARTELYRSL and ARFNDLRFV) and an analogue of a melanocyte autoantigen (ADAMTSL5, VRSRR-abu-LRL) implicated in psoriasis. These structures revealed that HLA-C*06:02 possesses a deep peptide-binding groove comprising two electronegative B- and E-pockets that coincide with the preference for P2 and P7 arginine anchors. The ADAMTSL5 autoantigen possessed a P7-Leu instead of the P7-Arg residue, but nevertheless was accommodated within the HLA-C*06:02 antigen-binding cleft. Collectively, our results provide the structural basis for understanding peptide repertoire selection in HLA-C*06:02.

Type I interferons (IFN-Is) are emerging as key drivers of inflammation and immunosuppression in chronic infection. Control of these infections requires IFN-I signaling; however, prolonged IFN-I signaling can lead to immune dysfunction. IFN-Is are also emerging as double-edged swords in cancer, providing necessary inflammatory signals, while initiating feedback suppression in both immune and cancer cells. Here, we review the proinflammatory and suppressive mechanisms potentiated by IFN-Is during chronic virus infections and discuss the similar, newly emerging dichotomy in cancer. We then discuss how this understanding is leading to new therapeutic concepts and immunotherapy combinations. We propose that, by modulating the immune response at its foundation, it may be possible to widely reshape immunity to control these chronic diseases.

OBJECTIVETo describe the investigation and control of a cluster ofSerratia marcescensbacteremia in a 505-bed tertiary-care center.METHODSCluster cases were defined as all patients withS. marcescensbacteremia between March 2 and April 7, 2014, who were found to have identical or related blood isolates determined by molecular typing with pulsed-field gel electrophoresis. Cases were compared using bivariate analysis with controls admitted at the same time and to the same service as the cases, in a 4:1 ratio.RESULTSIn total, 6 patients developedS. marcescensbacteremia within 48 hours after admission within the above period. Of these, 5 patients had identicalSerratiaisolates determined by molecular typing, and were included in a case-control study. Exposure to the post-anesthesia care unit was a risk factor identified in bivariate analysis. Evidence of tampered opioid-containing syringes on several hospital units was discovered soon after the initial cluster case presented, and a full narcotic diversion investigation was conducted. A nurse working in the post-anesthesia care unit was identified as the employee responsible for the drug diversion and was epidemiologically linked to all 5 patients in the cluster. No further cases were identified once the implicated employee’s job was terminated.CONCLUSIONIllicit drug use by healthcare workers remains an important mechanism for the development of bloodstream infections in hospitalized patients. Active mechanisms and systems should remain in place to prevent, detect, and control narcotic drug diversions and associated patient harm in the healthcare setting.Infect Control Hosp Epidemiol2017;38:1027–1031

The lymphoid tissue that drains the upper respiratory tract represents an important induction site for cytotoxic T lymphocyte (CTL) immunity to airborne pathogens and intranasal vaccines. Here, we investigated the role of the nasal-associated lymphoid tissues (NALTs), which are mucosal-associated lymphoid organs embedded in the submucosa of the nasal passage, in the initial priming and recall expansion of CD8+ T cells following an upper respiratory tract infection with a pathogenic influenza virus and immunization with a live attenuated influenza virus vaccine. Whereas NALTs served as the induction site for the recall expansion of memory CD8+ T cells following influenza virus infection or vaccination, they failed to support activation of naive CD8+ T cells. Strikingly, NALTs, unlike other lymphoid tissues, were not routinely surveyed during the steady state by circulating T cells. The selective recruitment of memory T cells into these lymphoid structures occurred in response to infection-induced elevation of the chemokine CXCL10, which attracted CXCR3+ memory CD8+ T cells. These results have significant implications for intranasal vaccines, which deliver antigen to mucosal-associated lymphoid tissue and aim to elicit protective CTL-mediated immunity.

General control nonderepressible 2 (GCN2) is an environmental sensor controlling transcription and translation in response to nutrient availability. Although GCN2 is a putative therapeutic target for immuno-oncology, its role in shaping the immune response to tumors is poorly understood. Here, we used mass cytometry, transcriptomics, and transcription factor-binding analysis to determine the functional impact of GCN2 on the myeloid phenotype and immune responses in melanoma. We found that myeloid-lineage deletion of GCN2 drives a shift in the phenotype of tumor-associated macrophages and myeloid-derived suppressor cells (MDSCs) that promotes antitumor immunity. Time-of-flight mass cytometry (CyTOF) and single-cell RNA sequencing showed that this was due to changes in the immune microenvironment with increased proinflammatory activation of macrophages and MDSCs and interferon-γ expression in intratumoral CD8+ T cells. Mechanistically, GCN2 altered myeloid function by promoting increased translation of the transcription factor CREB-2/ATF4, which was required for maturation and polarization of macrophages and MDSCs in both mice and humans, whereas targeting Atf4 by small interfering RNA knockdown reduced tumor growth. Last, analysis of patients with cutaneous melanoma showed that GCN2-dependent transcriptional signatures correlated with macrophage polarization, T cell infiltrates, and overall survival. Thus, these data reveal a previously unknown dependence of tumors on myeloid GCN2 signals for protection from immune attack.

Gamma delta (γδ) T cells are a highly heterogeneous population of lymphocytes that exhibit innate and adaptive immune properties. Despite comprising the majority of residing lymphocytes in many organs, the role of γδ T cells in transplantation outcomes is under‐researched. γδ T cells can recognise a diverse array of ligands and exert disparate effector functions. As such, they may potentially contribute to both allograft acceptance and rejection, as well as impacting on infection and post‐transplant malignancy. Here, we review the current literature on the role and function of γδ T cells following solid organ and hematopoietic stem cell transplantation.

Macleod et al. uncover how chronic viral infection modulates gastrointestinal immune and microbial composition to foster persistence and long-term disease susceptibilities. The authors also identify the central role of regulatory T cells to maintain viral sanctuary in the intestinal tract., Many pathogens subvert intestinal immunity to persist within the gastrointestinal tract (GIT); yet, the underlying mechanisms that enable sanctuary specifically in this reservoir are unclear. Using mass cytometry and network analysis, we demonstrate that chronic LCMV infection of the GIT leads to dysregulated microbial composition, a cascade of metabolic alterations, increased susceptibility to GI disease, and a system-wide recalibration of immune composition that defines viral persistence. Chronic infection led to outgrowth of activated Tbet–expressing T reg cell populations unique to the GIT and the rapid erosion of pathogen-specific CD8 tissue-resident memory T cells. Mechanistically, T reg cells and coinhibitory receptors maintained long-term viral sanctuary within the GIT, and their targeting reactivated T cells and eliminated this viral reservoir. Thus, our data provide a high-dimensional definition of the mechanisms of immune regulation that chronic viruses implement to exploit the unique microenvironment of the GIT and identify T reg cells as key modulators of viral persistence in the intestinal tract., Graphical Abstract

Background Full-length isoform quantification from RNA-Seq is a key goal in transcriptomics analyses and has been an area of active development since the beginning. The fundamental difficulty stems from the fact that RNA transcripts are long, while RNA-Seq reads are short. Results Here we use simulated benchmarking data that reflects many properties of real data, including polymorphisms, intron signal and non-uniform coverage, allowing for systematic comparative analyses of isoform quantification accuracy and its impact on differential expression analysis. Genome, transcriptome and pseudo alignment-based methods are included; and a simple approach is included as a baseline control. Conclusions Salmon, kallisto, RSEM, and Cufflinks exhibit the highest accuracy on idealized data, while on more realistic data they do not perform dramatically better than the simple approach. We determine the structural parameters with the greatest impact on quantification accuracy to be length and sequence compression complexity and not so much the number of isoforms. The effect of incomplete annotation on performance is also investigated. Overall, the tested methods show sufficient divergence from the truth to suggest that full-length isoform quantification and isoform level DE should still be employed selectively.

Despite current immunosuppressive strategies, long-term lung transplant outcomes remain poor due to rapid allogenic responses. Using a stringent mouse model of allo-airway transplantation, we identified the CCR4-ligand axis as a central node driving secondary lymphoid tissue homing and activation of the allogeneic T cells that prevent long-term allograft survival. CCR4 deficiency on transplant recipient T cells diminished allograft injury and when combined with CTLA4-Ig led to lung allograft accommodation lasting longer than in any previous study to our knowledge. Thus, we identify CCR4-ligand interactions as a central mechanism driving allogeneic transplant rejection and suggest it as a potential target to enhance long-term lung transplant survival.

Manipulation of the MHC-I presentation pathway, and thus limiting MHC-I cell surface expression, is used by many viruses to evade immune recognition. In particular, downregulation of MHC-I molecules at the cell surface can reduce the ability of CD8+ T cells to recognize viral peptides presented by MHC-I molecules and thereby delay viral clearance by CD8+ T cells. To date, MHC-I downregulation by influenza viruses has not been reported. Given that influenza virus infections are a global health concern and that CD8+ T cells play an important role in promoting influenza virus clearance and recovery from influenza disease, we investigated whether influenza A and B viruses (IAV, IBV) downregulated MHC-I as a novel mechanism to evade cellular immunity. Here, we showed that infection of several cell types, including epithelial A549 cells, with a panel of IAV and IBV viruses downregulated the surface MHC-I expression on IAV/IBV-infected cells during the late stages of influenza virus infection in vitro. This observation was consistent across a panel of class I-reduced (C1R) cell lines expressing 14 different HLA-A or -B alleles and a panel of 721.221 cell lines expressing 11 HLA-C alleles. Interestingly, IBV infection caused more pronounced reduction in surface MHC-I expression compared to IAV. Importantly, the two viruses utilized two distinct mechanisms for MHC-I downregulation. Our data demonstrated that while IAV caused a global loss of MHC-I within influenza-infected cells, IBV infection resulted in the preferential loss of MHC-I molecules from the cell surface, consequent of delayed MHC-I trafficking to the cell surface, resulting from retaining MHC-I intracellularly during IBV infection. Overall, our study suggests that influenza viruses across both IAV and IBV subtypes have the potential to downregulate MHC-I surface expression levels. Our findings provide new insights into the host-pathogen interaction of influenza A and B viruses and inform the design of novel vaccine strategies against influenza viruses.

Flow cytometry is a widely applied approach for exploratory immune profiling and biomarker discovery in cancer and other diseases. However, flow cytometry is limited by the number of parameters that can be simultaneously analyzed, severely restricting its utility. Recently, the advent of mass cytometry (CyTOF) has enabled high dimensional and unbiased examination of the immune system, allowing simultaneous interrogation of a large number of parameters. This is important for deep interrogation of immune responses and particularly when sample sizes are limited (such as in tumors). Our goal was to compare the accuracy and reproducibility of CyTOF against flow cytometry as a reliable analytic tool for human PBMC and tumor tissues for cancer clinical trials. We developed a 40+ parameter CyTOF panel and demonstrate that compared to flow cytometry, CyTOF yields analogous quantification of cell lineages in conjunction with markers of cell differentiation, function, activation, and exhaustion for use with fresh and viably frozen PBMC or tumor tissues. Further, we provide a protocol that enables reliable quantification by CyTOF down to low numbers of input human cells, an approach that is particularly important when cell numbers are limiting. Thus, we validate CyTOF as an accurate approach to perform high dimensional analysis in human tumor tissue and to utilize low cell numbers for subsequent immunologic studies and cancer clinical trials.

Airway epithelial cells and macrophages differ markedly in their responses to influenza A virus (IAV) infection. To investigate transcriptional responses underlying these differences, purified subsets of type II airway epithelial cells (ATII) and alveolar macrophages (AM) recovered from the lungs of mock- or IAV-infected mice at 9 h postinfection were subjected to RNA sequencing. This time point was chosen to allow for characterization of cell types first infected with the virus inoculum, prior to multicycle virus replication and the infiltration of inflammatory cells into the airways. In the absence of infection, AM predominantly expressed genes related to immunity, whereas ATII expressed genes consistent with their physiological roles in the lung. Following IAV infection, AM almost exclusively activated cell-intrinsic antiviral pathways that were dependent on interferon (IFN) regulatory factor 3/7 (IRF3/7) and/or type I IFN signaling. In contrast, IAV-infected ATII activated a broader range of physiological responses, including cell-intrinsic antiviral pathways, which were both independent of and dependent on IRF3/7 and/or type I IFN. These data suggest that transcriptional profiles hardwired during development are a major determinant underlying the different responses of ATII and AM to IAV infection. IMPORTANCE Airway epithelial cells (AEC) and airway macrophages (AM) represent major targets of influenza A virus (IAV) infection in the lung, yet the two cell types respond very differently to IAV infection. We have used RNA sequencing to define the host transcriptional responses in each cell type under steady-state conditions as well as following IAV infection. To do this, different cell subsets isolated from the lungs of mock- and IAV-infected mice were subjected to RNA sequencing. Under steady-state conditions, AM and AEC express distinct transcriptional activities, consistent with distinct physiological roles in the airways. Not surprisingly, these cells also exhibited major differences in transcriptional responses following IAV infection. These studies shed light on how the different transcriptional architectures of airway cells from two different lineages drive transcriptional responses to IAV infection.

Summary Chronic viral infections increase severity of Mycobacterium tuberculosis (Mtb) coinfection. Here, we examined how chronic viral infections alter the pulmonary microenvironment to foster coinfection and worsen disease severity. We developed a coordinated system of chronic virus and Mtb infection that induced central clinical manifestations of coinfection, including increased Mtb burden, extra-pulmonary dissemination, and heightened mortality. These disease states were not due to chronic virus-induced immunosuppression or exhaustion; rather, increased amounts of the cytokine TNFα initially arrested pulmonary Mtb growth, impeding dendritic cell mediated antigen transportation to the lymph node and subverting immune-surveillance, allowing bacterial sanctuary. The cryptic Mtb replication delayed CD4 T cell priming, redirecting T helper (Th) 1 toward Th17 differentiation and increasing pulmonary neutrophilia, which diminished long-term survival. Temporally restoring CD4 T cell induction overcame these diverse disease sequelae to enhance Mtb control. Thus, Mtb co-opts TNFα from the chronic inflammatory environment to subvert immune-surveillance, avert early immune function, and foster long-term coinfection.

Marine sediments from coastal Florida, USA impacted by Karenia brevis blooms were resuspended in seawater and irradiated in a solar simulator to determine if sedimentary bound PbTx-2 is photolytically released into the aqueous phase. All bulk and size-fractionated (

In order to assess the use of Spanish moss (Tillandsia usneoides L.) as a biomonitor for atmospheric reactive nitrogen (Nr), T. usneoides samples were collected in urban Wilmington, NC, USA and at a remote barrier island 30 km away (Bald Head Island, NC, USA), and were analyzed for N content and δ15N. The mean urban N content (n = 64) was 0.8 ± 0.5 % whereas the island samples (n = 4) mean was 0.1 ± 0.04 %. δ15N values for Wilmington samples ranged from −13.9 to +23.6‰ (mean = −2.2 ± 9.3‰) while δ15N for island samples (n = 4) ranged from −12.4 to −9.8‰ (mean = −11.2 ± 1.1‰). Both the N content and δ15N values in the urban air shed were significantly higher than those at the barrier island due to urban anthropogenic Nr emissions including inputs from vehicles. δ15N of T. usneoides was correlated with road density, and δ15N values at a road transect decreased with distance from the road, indicating the importance of vehicle emissions as a source of Nr to urban ecosystems. A 3.5 kg ha−1y−1 bulk N deposition rate was estimated for the air shed using average T. usneoides N content (0.8 %) and a previously developed model relating N content in European moss from 16 countries to bulk N deposition. This deposition is remarkably similar to the N deposition rate (3.19 kg ha−1y−1) measured by the U.S. EPA Clean Air Status and Trends Network at the nearest coastal collection site. This suggests the relationship between European moss N content and bulk N deposition is also true for T. usneoides in the southeastern USA and underscores the efficacy of this method. Results presented here are significant because they indicate that T. usneoides can be used as an effective and inexpensive biomonitor for atmospheric N deposition.

It is well established that glycosaminoglycans (GAGs) function as attachment factors for human metapneumovirus (HMPV), concentrating virions at the cell surface to promote interaction with other receptors for virus entry and infection. There is increasing evidence to suggest that multiple receptors may exhibit the capacity to promote infectious entry of HMPV into host cells; however, definitive identification of specific transmembrane receptors for HMPV attachment and entry is complicated by the widespread expression of cell surface GAGs. pgsA745 Chinese hamster ovary (CHO) cells are deficient in the expression of cell surface GAGs and resistant to HMPV infection. Here, we demonstrate that the expression of the Ca 2+ -dependent C-type lectin receptor (CLR) DC-SIGN (CD209L) or L-SIGN (CD209L) rendered pgsA745 cells permissive to HMPV infection. Unlike infection of parental CHO cells, HMPV infection of pgsA745 cells expressing DC-SIGN or L-SIGN was dynamin dependent and inhibited by mannan but not by pretreatment with bacterial heparinase. Parental CHO cells expressing DC-SIGN/L-SIGN also showed enhanced susceptibility to dynamin-dependent HMPV infection, confirming that CLRs can promote HMPV infection in the presence or absence of GAGs. Comparison of pgsA745 cells expressing wild-type and endocytosis-defective mutants of DC-SIGN/L-SIGN indicated that the endocytic function of CLRs was not essential but could contribute to HMPV infection of GAG-deficient cells. Together, these studies confirm a role for CLRs as attachment factors and entry receptors for HMPV infection. Moreover, they define an experimental system that can be exploited to identify transmembrane receptors and entry pathways where permissivity to HMPV infection can be rescued following the expression of a single cell surface receptor. IMPORTANCE On the surface of CHO cells, glycosaminoglycans (GAGs) function as the major attachment factor for human metapneumoviruses (HMPV), promoting dynamin-independent infection. Consistent with this, GAG-deficient pgaA745 CHO cells are resistant to HMPV. However, expression of DC-SIGN or L-SIGN rendered pgsA745 cells permissive to dynamin-dependent infection by HMPV, although the endocytic function of DC-SIGN/L-SIGN was not essential for, but could contribute to, enhanced infection. These studies provide direct evidence implicating DC-SIGN/L-SIGN as an alternate attachment factor for HMPV attachment, promoting dynamin-dependent infection via other unknown receptors in the absence of GAGs. Moreover, we describe a unique experimental system for the assessment of putative attachment and entry receptors for HMPV.

Many insights regarding the pathogenesis of human influenza A virus (IAV) infections have come from studies in mice and ferrets. Surfactant protein (SP)-D is the major neutralizing inhibitor of IAV in mouse airway fluids and SP-D-resistant IAV mutants show enhanced virus replication and virulence in mice. Herein, we demonstrate that sialylated glycoproteins, rather than SP-D, represent the major neutralizing inhibitors against H3 subtype viruses in airway fluids from naïve ferrets. Moreover, while resistance to neutralizing inhibitors is a critical factor in modulating virus replication and disease in the mouse model, it does not appear to be so in the ferret model, as H3 mutants resistant to either SP-D or sialylated glycoproteins in ferret airway fluids did not show enhanced virulence in ferrets. These data have important implications for our understanding of pathogenesis and immunity to human IAV infections in these two widely used animal models of infection.

BACKGROUND Ragweed frequently causes seasonal allergies in North America and Europe. In the United States, several related ragweed species with diverse geographical distribution cause allergic symptoms. Cross-reactivity towards related ragweed species of IgE and treatment-induced IgG4 has been demonstrated previously. However, less is known about the underlying T-cell cross-reactivity. METHODS The allergen content of ragweed extracts was determined by mass spectrometry and related to T-cell epitopes of Amb a allergens (group 1, 3, 4, 5, 8 and 11) in 20 American ragweed allergic patients determined by FluoroSpot and proliferation assays. T-cell responses to 50 frequently recognized Amb a-derived T-cell epitopes and homologous peptides from western ragweed (Amb p), giant ragweed (Amb t) and mugwort (Art v) were investigated in an additional 11 American and 14 Slovakian ragweed allergic donors. RESULTS Ragweed extracts contained all known allergens and isoallergens thereof. Donor T-cell responses were diverse and directed against all Amb a 1 isoallergens and to most minor allergens investigated. Similar response patterns were seen in American and Slovakia donors. Several epitopes were cross-reactive between isoallergens and ragweed species, some even including mugwort. T-cell cross-reactivity generally correlated with allergen sequence homology. CONCLUSION T-cell epitopes of multiple allergens/isoallergens are involved in the diverse T-cell responses in ragweed allergic individuals. T-cell lines were highly cross-reactive to epitopes of related ragweed species without any apparent geographical response bias. These data support that different ragweed species can be considered an allergen homology group with Amb a as the representative species regarding diagnosis as well as allergy immunotherapy.

About one-third of dilated cardiomyopathy (DCM) cases are caused by mutations in sarcomere or cytoskeletal proteins. However, treating the cytoskeleton directly is not possible because drugs that bind to actin are not well tolerated. Mutations in the actin binding protein CAP2 can cause DCM and KO mice, either whole body (CAP2-KO) or cardiomyocyte-specific KOs (CAP2-CKO) develop DCM with cardiac conduction disease. RNA sequencing analysis of CAP2-KO hearts and isolated cardiomyocytes revealed overactivation of fetal genes, including serum response factor–regulated (SRF-regulated) genes such as Myl9 and Acta2 prior to the emergence of cardiac disease. To test if we could treat CAP2-KO mice, we synthesized and tested the SRF inhibitor CCG-1423-8u. CCG-1423-8u reduced expression of the SRF targets Myl9 and Acta2, as well as the biomarker of heart failure, Nppa. The median survival of CAP2-CKO mice was 98 days, while CCG-1423-8u–treated CKO mice survived for 116 days and also maintained normal cardiac function longer. These results suggest that some forms of sudden cardiac death and cardiac conduction disease are under cytoskeletal stress and that inhibiting signaling through SRF may benefit DCM by reducing cytoskeletal stress.

Purpose The long-term success of lung transplantation (LTx) is limited by CLAD. The complement-independent pathway of ABMR, driven by antibody-dependent cellular cytotoxicity (ADCC) may contribute to CLAD. ADCC is mediated through the CD16 Fc receptor expressed on NK cells, however a role for different NK cell subsets in mediating ABMR is yet to be elucidated. In this pilot study, we investigated whether the presence of donor-specific antibodies (DSA) following LTx effects NK cell ADCC and whether particular NK cell subsets exhibited superior ADCC activity. Methods Peripheral blood mononuclear cells (PBMC) were isolated from LTx recipients at 3 and 12-months post-LTx. NK cell ADCC activity (degranulation (CD107a) and the production of cytokines from PBMC (interferon gamma (IFN-γ) and tumor necrosis alpha (TNF-α))) was measured by flow cytometry following antibody-dependent activation (ADA). The detection of anti-human leukocyte antigen (HLA) antibodies at the same time points was determined by Luminex. LTx recipients were classified as DSA positive (DSA+, presence of anti-donor HLA antibodies, n=12), anti-HLA antibody positive (HLA+, presence of anti-HLA antibodies but not to donor HLA, n=12) or no HLA antibodies (Ab-, no anti-HLA antibodies detected, n=7). Results Surprisingly, a higher proportion of NK cells expressed the activating receptor NKG2C in the DSA+ group (p=0.0078). Furthermore, NKG2C+ NK cells isolated from recipients with HLA antibodies were significantly more activated than NKG2A+ cells. Following ADA, a higher proportion of NKG2C+ NK cells produced cytokines in DSA+ (IFNγ, p=0.0095) and HLA+ (IFNγ: p=0.0317; TNFα: p=0.0159) recipients, compared to NKG2A+ cells. In addition, CD16 expression was more down-regulated following ADA in DSA+ recipients (p=0.0084). Conclusion Our data shows that the presence of DSA impacts on the phenotype and function of NK cells following LTx. Moreover, as previous research has shown an association between NKG2C expression, cytomegalovirus (CMV) infection and CLAD, our study potentially provides a missing link between CMV infection and CLAD. Future confirmation of NKG2C+ NK cells as mediators of ABMR will lead to development of targeted diagnostic criteria and specific therapies to offer early therapeutic interventions to prevent CLAD, thereby improving LTx outcomes.

Previous studies demonstrated that oral antibiotic (ABX) treatment prior to and during influenza A virus (IAV) infection of adult mice profoundly altered gut microbiota (GM) and was associated with increased susceptibility and impaired immunity to IAV. We examined the impact of ABX during critical times relevant to the establishment of GM in early life (using perinatal treatment of neonates and direct treatment of juvenile mice) and asked whether cessation of ABX treatment in early life had lasting effects on GM composition and anti-IAV immunity. ABX treatment significantly changed GM composition in juvenile mice and in ABX-treated dams. However, if ABX treatment ceased at the time of infection, neither neonates nor juvenile mice showed enhanced susceptibility to IAV, nor were major differences detected in cellular and humoral adaptive antiviral immunity. Thus, while ABX treatment alters GM diversity in early life, cessation and subsequent re-colonization correlates with effective immunity against IAV.

Cytotoxic T lymphocytes (CTL) kill target cells on the basis of cell-surface antigen recognition and are important in the host response to tumors, transplants, and viruses. This unit presents several protocols for generating and measuring CTL activity. The first describes generating CTL against some of the most commonly used target antigens. Two methods for the quantitation of CTL activity are described based on the two pathways used bt CTL to kill target cells. In one pathway, they release lytic granules containing perforin and granzymes, leading to apoptosis and target cell lysis. In a second pathway, they trigger apoptosis via Fas/Fas ligand interactions. In the chromium-release assay provided here, labeled antigenic targets are recognized and lysed, releasing radioactivity into the supernatant. In the JAM test protocol, CTL activity is determined by measuring degradation of radioactively labeled DNA in target cells that have undergone apoptotic cell death. Rather than measuring release of radioactivity, the JAM test measures the amount of DNA retained in target cells that are not killed by CTL. Two support protocols detail the generation of CTL precursors (CTLp) against antigens that require priming in vivo. A second set of support protocols describe the preparation of both stimulator and target cells for these responses using two representative antigens, trinitrophenyl and viruses. Finally, two alternate protocols illustrate how to determine total CTLp activity in a population that might express cytolytic activity. These protocols bypass MHC restriction and the original antigen specificity of CTLp by polyclonal stimulation of CTLp with mitogens followed by attachment of CTL to target cells and subsequent cytolysis.