Semiquantitative Proteomics Enables Mapping of Murine Neutrophil Dynamics following Lethal Influenza Virus Infection.

Neutrophils are rapidly deployed innate immune cells, and excessive recruitment is causally associated with influenza-induced pathologic conditions. Despite this, the complete set of influenza lethality-associated neutrophil effector proteins is currently unknown. Whether the expression of these proteins is predetermined during bone marrow (BM) neutrophil maturation or further modulated by tissue compartment transitions has also not been comprehensively characterized at a proteome-wide scale. In this study, we used high-resolution mass spectrometry to map how the proteomes of murine neutrophils change comparatively across BM, blood, and the alveolar airspaces to deploy an influenza lethality-associated response. Following lethal influenza infection, mature neutrophils undergo two infection-dependent and one context-independent compartmental transitions. Translation of type I IFN-stimulated genes is first elevated in the BM, preceding the context-independent downregulation of ribosomal proteins observed in blood neutrophils. Following alveolar airspace infiltration, the bronchoalveolar lavage (BAL) neutrophil proteome is further characterized by a limited increase in type I IFN-stimulated and metal-sequestering proteins as well as a decrease in degranulation-associated proteins. An influenza-selective and dose-dependent increase in antiviral and lipid metabolism-associated proteins was also observed in BAL neutrophils, indicative of a modest capacity for pathogen response tuning. Altogether, our study provides new and comprehensive evidence that the BAL neutrophil proteome is shaped by BM neutrophil maturation as well as subsequent compartmental transitions following lethal influenza infection.

Influenza A Virus Infection Induces Viral and Cellular Defective Ribosomal Products Encoded by Alternative Reading Frames.

The importance of antiviral CD8+ T cell recognition of alternative reading frame (ARF)-derived peptides is uncertain. In this study, we describe an epitope (NS1-ARF21-8) present in a predicted 14-residue peptide encoded by the +1 register of NS1 mRNA in the influenza A virus (IAV). NS1-ARF21-8 elicits a robust, highly functional CD8+ T cell response in IAV-infected BALB/c mice. NS1-ARF21-8 is presented from unspliced NS mRNA, likely from downstream initiation on a Met residue that comprises the P1 position of NS1-ARF21-8 Derived from a 14-residue peptide with no apparent biological function and negligible impacts on IAV infection, infectivity, and pathogenicity, NS1-ARF21-8 provides a clear demonstration of how immunosurveillance exploits natural errors in protein translation to provide antiviral immunity. We further show that IAV infection enhances a model cellular ARF translation, which potentially has important implications for virus-induced autoimmunity.

Host CD8α+ and CD103+ dendritic cells prime transplant antigen-specific CD8+ T cells via cross-dressing.

The participation of dendritic cells (DCs) in CD8+ T-cell-mediated allograft rejection is a long-standing question of great clinical relevance for tissue transplantation. Here, we show that Batf3-/- mice demonstrate delayed allo-skin graft rejection and are deficient in priming allo-specific CD8+ T cells. Batf3-/- mouse priming is restored by transferring either purified CD8α+ or CD103+ DCs, demonstrating the critical role of these cells in alloreactivity. Using Db -restricted antiviral F5 transgenic T-cell receptor T cells, which we demonstrate are alloreactive with H-2Kd , we show that cross-dressing of CD8α+ and CD103+ primes CD8+ T-cell or allo-specific responses in vitro and in vivo. These findings suggest novel strategies for moderating tissue rejection based on interfering with DC cross-dressing or subsequent interaction with T cells.

CXCR4+ cells are increased in lung tissue of patients with idiopathic pulmonary fibrosis.

BACKGROUND: CXCR4, a transmembrane-receptor located on epithelial cells that is activated by CXCL12, may have a role in IPF via migration of CXCR4+ fibrocytes to the lung. However, its expression has not been fully characterised in idiopathic pulmonary fibrosis (IPF) or other fibrotic interstitial lung diseases (ILDs). CXCL12 is constitutively expressed in the bone marrow, and levels of CXCR4 regulate control of this signalling pathway. The aim of this study was to profile the expression of CXCR4 in lung tissue and peripheral circulation of patients with IPF and other fibrotic ILDs. METHODS: Expression of CXCR4 on peripheral blood mononuclear cells (PBMCs) was examined by flow cytometry in 20 patients with IPF and 10 age-matched non-disease control (NDC) donors. Levels of CXCL12 in human plasma were measured by ELISA. Expression of CXCR4, CXCL12, CD45, and e-cadherin was assessed in IPF (n = 10), other fibrotic ILD (n = 8) and NDC (n = 10) lung tissue by multiplex immunohistochemistry (OPAL) and slides were scanned using a Vectra 3 scanner. Cells were quantified with computer automated histological analysis software (HALO). RESULTS: In blood, the number of CXCR4+ cells was lower but the level of CXCL12 was higher in patients with IPF compared to NDC donors. Elevated CXCR4 expression was detected in lung tissue from patients with IPF and other fibrotic ILDs compared to NDC. There were higher levels of CXCR4+/e-cadherin+/CXCL12+ (epithelial) cells in IPF lung tissue compared to NDC, but there was no difference in the numbers of CXCR4+/CD45+/CXCL12+ (myeloid) cells between the two groups. CONCLUSIONS: This report demonstrates that CXCR4 is overexpressed not only in IPF but also in other ILDs and expression is particularly prominent within both honeycomb cysts and distal airway epithelium. This observation supports the hypothesis that CXCR4 may drive tissue fibrosis through binding its specific ligand CXCL12. Although CXCR4 expressing cells could be either of epithelial or myeloid origin it appears that the former is more prominent in IPF lung tissue. Further characterization of the cells of the honeycomb cyst may lead to a better understanding of the fibrogenic processes in IPF and other end-stage fibrotic ILDs.

LMP2 immunoproteasome promotes lymphocyte survival by degrading apoptotic BH3-only proteins.

The role of the immunoproteasome is perceived as confined to adaptive immune responses given its ability to produce peptides ideal for MHC Class-I binding. Here, we demonstrate that the immunoproteasome subunit, LMP2, has functions beyond its immunomodulatory role. Using LMP2-deficient mice, we demonstrate that LMP2 is crucial for lymphocyte development and survival in the periphery. Moreover, LMP2-deficient lymphocytes show impaired degradation of key BH3-only proteins, resulting in elevated levels of pro-apoptotic BIM and increased cell death. Interestingly, LMP2 is the sole immunoproteasome subunit required for BIM degradation. Together, our results suggest LMP2 has important housekeeping functions and represents a viable therapeutic target for cancer.

Increasing viral dose causes a reversal in CD8+ T cell immunodominance during primary influenza infection due to differences in antigen presentation, T cell avidity, and precursor numbers.

T cell responses are characterized by the phenomenon of immunodominance (ID), whereby peptide-specific T cells are elicited in a reproducible hierarchy of dominant and subdominant responses. However, the mechanisms that give rise to ID are not well understood. We investigated the effect of viral dose on primary CD8(+) T cell (T(CD8+)) ID by injecting mice i.p. with various doses of influenza A virus and assessing the primary T(CD8+) response to five dominant and subdominant peptides. Increasing viral dose enhanced the overall strength of the T(CD8+) response, and it altered the ID hierarchy: specifically, NP(366-374) T(CD8+) were dominant at low viral doses but were supplanted by PA(224-233) T(CD8+) at high doses. To understand the basis for this reversal, we mathematically modeled these T(CD8+) responses and used Bayesian statistics to obtain estimates for Ag presentation, T(CD8+) precursor numbers, and avidity. Interestingly, at low viral doses, Ag presentation most critically shaped ID hierarchy, enabling T(CD8+) specific to the more abundantly presented NP(366-374) to dominate. By comparison, at high viral doses, T(CD8+) avidity and precursor numbers appeared to be the major influences on ID hierarchy, resulting in PA(224-233) T(CD8+) usurping NP(366-374) cells as the result of higher avidity and precursor numbers. These results demonstrate that the nature of primary T(CD8+) responses to influenza A virus is highly influenced by Ag dose, which, in turn, determines the relative importance of Ag presentation, T(CD8+) avidity, and precursor numbers in shaping the ID hierarchy. These findings provide valuable insights for future T(CD8+)-based vaccination strategies.

CRIMP: a CRISPR/Cas9 insertional mutagenesis protocol and toolkit.

Site-directed insertion is a powerful approach for generating mutant alleles, but low efficiency and the need for customisation for each target has limited its application. To overcome this, we developed a highly efficient targeted insertional mutagenesis system, CRIMP, and an associated plasmid toolkit, CRIMPkit, that disrupts native gene expression by inducing complete transcriptional termination, generating null mutant alleles without inducing genetic compensation. The protocol results in a high frequency of integration events and can generate very early targeted insertions, during the first cell division, producing embryos with expression in one or both halves of the body plan. Fluorescent readout of integration events facilitates selection of successfully mutagenized fish and, subsequently, visual identification of heterozygous and mutant animals. Together, these advances greatly improve the efficacy of generating and studying mutant lines. The CRIMPkit contains 24 ready-to-use plasmid vectors to allow easy and complete mutagenesis of any gene in any reading frame without requiring custom sequences, modification, or subcloning.

Optimal conditions required for influenza A infection-enhanced cross-priming of CD8⁺ T cells specific to cell-associated antigens.

Dendritic cells can take up exogenous tumor antigens and present their antigenic epitopes to CD8⁺ T cells (T(CD8⁺)), a process called cross-presentation. Cross-presentation is especially important in antitumor immunity because tumor cells, although carrying tumor antigens, do not activate naive T cells efficiently because of a lack of co-stimulatory molecules. Our group has recently shown that influenza A virus (IAV) infection of allogeneic cells lead to enhanced cross-priming of T(CD8⁺) specific to cellular antigens. To develop this into a potential vaccine strategy, in this study, we have systematically investigated the numbers of allogeneic cells infected by IAV, IAV doses and their infectious activity, the length of in vitro infection and other associated factors. We have defined the optimal immune-enhancing conditions and we have also shown in vivo that such enhanced cross-priming did lead to enhanced tumor protection. The knowledge should be useful for developing more robust cancer vaccine.

Influenza A virus infection-induced macroautophagy facilitates MHC class II-restricted endogenous presentation of an immunodominant viral epitope.

CD4+ T cells recognize peptides presented by major histocompatibility complex class II molecules (MHC-II). These peptides are generally derived from exogenous antigens. Macroautophagy has been reported to promote endogenous antigen presentation in viral infections. However, whether influenza A virus (IAV) infection-induced macroautophagy also leads to endogenous antigen presentation through MHC-II is still debated. In this study, we show that IAV infection leads to endogenous presentation of an immunodominant viral epitope NP311-325 by MHC-II to CD4+ T cells. Mechanistically, such MHC-II-restricted endogenous IAV antigen presentation requires de novo protein synthesis as it is inhibited by the protein synthesis inhibitor cycloheximide, and a functional ER-Golgi network as it is totally blocked by Brefeldin A. These results indicate that MHC-II-restricted endogenous IAV antigen presentation is dependent on de novo antigen and/or MHC-II synthesis, and transportation through the ER-Golgi network. Furthermore, such endogenous IAV antigen presentation by MHC-II is enhanced by TAP deficiency, indicating some antigenic peptides are of cytosolic origin. Most importantly, the bulk of such MHC-II-restricted endogenous IAV antigen presentation is blocked by autophagy inhibitors (3-MA and E64d) and deletion of autophagy-related genes, such as Beclin1 and Atg7. We have further demonstrated that in dendritic cells, IAV infection prevents autophagosome-lysosome fusion and promotes autophagosome fusion with MHC class II compartment (MIIC), which likely promotes endogenous IAV antigen presentation by MHC-II. Our results provide strong evidence that IAV infection-induced autophagosome formation facilitates endogenous IAV antigen presentation by MHC-II to CD4+ T cells. The implication for influenza vaccine design is discussed.

Monocyte apoptotic bodies are vehicles for influenza A virus propagation.

The disassembly of apoptotic cells into small membrane-bound vesicles termed apoptotic bodies (ApoBDs) is a hallmark of apoptosis; however, the functional significance of this process is not well defined. We recently discovered a new membrane protrusion (termed beaded apoptopodia) generated by apoptotic monocytes which fragments to release an abundance of ApoBDs. To investigate the function of apoptotic monocyte disassembly, we used influenza A virus (IAV) infection as a proof-of-concept model, as IAV commonly infects monocytes in physiological settings. We show that ApoBDs generated from IAV-infected monocytes contained IAV mRNA, protein and virions and consequently, could facilitate viral propagation in vitro and in vivo, and induce a robust antiviral immune response. We also identified an antipsychotic, Haloperidol, as an unexpected inhibitor of monocyte cell disassembly which could impair ApoBD-mediated viral propagation under in vitro conditions. Together, this study reveals a previously unrecognised function of apoptotic monocyte disassembly in the pathogenesis of IAV infections.

Broad-Based CD4+ T Cell Responses to Influenza A Virus in a Healthy Individual Who Lacks Typical Immunodominance Hierarchy.

Influenza A virus (IAV) infection is a significant cause of morbidity and mortality worldwide. CD4+ T cell responses have been shown to be important for influenza protection in mouse models and in human volunteers. IAV antigen-specific CD4+ T cell responses were found to focus on matrix 1 (M1) and nucleoprotein (NP) at the protein antigen level. At the epitope level, only several epitopes within M1 and NP were recognized by CD4+ T cells. And the epitope-specific CD4+ T cell responses showed a typical immunodominance hierarchy in most of the healthy individuals studied. In this study, we reported one case of atypical immunodominance hierarchy of CD4+ T cell responses to IAV. M1 and NP were still the immunodominant targets of CD4+ T cell responses. However, CD4+ T cell responses specific to 11 epitopes derived from M1 and NP were detected and showed no significant immunodominance hierarchy. Such an atypical pattern is likely determined by the individual's HLA alleles. These findings will help us better understand the anti-IAV immunity as a whole and improve future vaccines against IAV.

An optimized method for establishing high purity murine CD8+ T cell cultures.

Establishing CD8(+) T cell cultures has been empirical and the published methods have been largely individual laboratory based. In this study, we optimized culturing conditions and show that IL-2 concentration is the most critical factor for the success of establishing CD8(+) T cell cultures. High IL-2 concentration encouraged T cells to non-specifically proliferate, express a B cell marker, B220, and undergo apoptosis. These cells also lose typical irregular T cell morphology and are incapable of sustaining long-term cultures. Using tetramer and intracellular cytokine assessments, we further demonstrated that many antigen-specific T cells have been rendered nonfunctional when expanded under high IL-2 concentration. When IL-2 is used in the correct range, B220-mediated cell depletion greatly enhanced the success rate of such T cell cultures.

Fine-mapping naturally occurring NY-ESO-1 antibody epitopes in melanoma patients' sera using short overlapping peptides and full-length recombinant protein.

The tumor antigen NY-ESO-1 is one of the most antigenic cancer-testis antigens, first identified by serologic analysis of a recombinant cDNA expression library (SEREX). NY-ESO-1 is expressed in different types of cancers including melanoma. NY-ESO-1-specific spontaneous humoral and cellular immune responses are detected in a large proportion of patients with advanced NY-ESO-1-expressing cancers. Therefore NY-ESO-1 is a good candidate antigen for immunotherapy. Although cellular immune responses to NY-ESO-1 are well characterized, much less is known about the humoral immune responses. In this study, we finely mapped linear antibody epitopes using sera from melanoma patients and shorter overlapping peptide sets. We have shown that melanoma patients' humoral immune systems responded to NY-ESO-1 differently in each individual with widely differing antibody specificity, intensity and antibody subtypes. This knowledge will help us further understand anti-tumor immunity and may also help us to monitor cancer progress and cancer vaccine efficacy in the future.

Resident CD8(+) and migratory CD103(+) dendritic cells control CD8 T cell immunity during acute influenza infection.

The identification of the specific DC subsets providing a critical role in presenting influenza antigens to naïve T cell precursors remains contentious and under considerable debate. Here we show that CD8(+) T lymphocyte (TCD8+) responses are severely hampered in C57BL/6 mice deficient in the transcription factor Batf3 after intranasal challenge with influenza A virus (IAV). This transcription factor is required for the development of lymph node resident CD8(+) and migratory CD103(+)CD11b(-) DCs and we found both of these subtypes could efficiently stimulate anti-IAV TCD8+. Using a similar ex vivo approach, many publications on this subject matter excluded a role for resident, non-migratory CD8(+) DC. We postulate the differences reported can partially be explained by how DC are phenotyped, namely the use of MHC class II to segregate subtypes. Our results show that resident CD8(+) DC upregulate this marker during IAV infection and we advise against its use when isolating DC subtypes.

A novel HLA-B18 restricted CD8+ T cell epitope is efficiently cross-presented by dendritic cells from soluble tumor antigen.

NY-ESO-1 has been a major target of many immunotherapy trials because it is expressed by various cancers and is highly immunogenic. In this study, we have identified a novel HLA-B*1801-restricted CD8(+) T cell epitope, NY-ESO-1(88-96) (LEFYLAMPF) and compared its direct- and cross-presentation to that of the reported NY-ESO-1(157-165) epitope restricted to HLA-A*0201. Although both epitopes were readily cross-presented by DCs exposed to various forms of full-length NY-ESO-1 antigen, remarkably NY-ESO-1(88-96) is much more efficiently cross-presented from the soluble form, than NY-ESO-1(157-165). On the other hand, NY-ESO-1(157-165) is efficiently presented by NY-ESO-1-expressing tumor cells and its presentation was not enhanced by IFN-γ treatment, which induced immunoproteasome as demonstrated by Western blots and functionally a decreased presentation of Melan A(26-35); whereas NY-ESO-1(88-96) was very inefficiently presented by the same tumor cell lines, except for one that expressed high level of immunoproteasome. It was only presented when the tumor cells were first IFN-γ treated, followed by infection with recombinant vaccinia virus encoding NY-ESO-1, which dramatically increased NY-ESO-1 expression. These data indicate that the presentation of NY-ESO-1(88-96) is immunoproteasome dependent. Furthermore, a survey was conducted on multiple samples collected from HLA-B18(+) melanoma patients. Surprisingly, all the detectable responses to NY-ESO-1(88-96) from patients, including those who received NY-ESO-1 ISCOMATRIX™ vaccine were induced spontaneously. Taken together, these results imply that some epitopes can be inefficiently presented by tumor cells although the corresponding CD8(+) T cell responses are efficiently primed in vivo by DCs cross-presenting these epitopes. The potential implications for cancer vaccine strategies are further discussed.

DNA vaccine encoding avian influenza virus H5 and Esat-6 of Mycobacterium tuberculosis improved antibody responses against AIV in chickens.

The H5 gene of avian influenza virus (AIV) strain A/chicken/Malaysia/5744/2004(H5N1) was cloned into pcDNA3.1 vector, and Esat-6 gene of Mycobacterium tuberculosis was fused into downstream of the H5 gene as a genetic adjuvant for DNA vaccine candidates. The antibody level against AIV was measured using enzyme-linked immunosorbent assay (ELISA) and haemagglutination inhibition (HI) test. Sera obtained from specific-pathogen-free chickens immunized with pcDNA3.1/H5 and pcDNA3.1/H5/Esat-6 demonstrated antibody responses as early as 2 weeks after the first immunization. Furthermore, the overall HI antibody titer in chickens immunized with pcDNA3.1/H5/Esat-6 was higher compared to the chickens immunized with pcDNA3.1/H5 (p<0.05). The results suggested that Esat-6 gene of M. tuberculosis is a potential genetic adjuvant for the development of effective H5 DNA vaccine in chickens.