SP-R210 isoforms of Myosin18A modulate endosomal sorting and recognition of influenza A virus infection in macrophages.

Influenza A virus (IAV) infection causes acute and often lethal inflammation in the lung. The role of macrophages in this adverse inflammation is partially understood. The surfactant protein A receptor 210 (SP-R210) consists of two isoforms, a long (L) SP-R210L and a short (S) SP-R210S isoform encoded by alternative splicing of the myosin 18A gene. We reported that disruption of SP-R210L enhances cytosolic and endosomal antiviral response pathways. Here, we report that SP-R210L antagonizes type I interferon ß (IFNß), as depletion of SP-R210L potentiates IFNß secretion. SP-R210 antibodies enhance and attenuate IFNß secretion in SP-R210L replete and deficient macrophages, respectively, indicating that SP-R210 isoform stoichiometry alters macrophage function intrinsically. This reciprocal response is coupled to unopposed and restricted expression of viral genes in control and SP-R210L-deficient macrophages, respectively. Human monocytic cells with sub-stoichiometric expression of SP-R210L resist IAV infection, whereas alveolar macrophages with increased abundance of SP-R210L permit viral gene expression similar to murine macrophages. Uptake and membrane binding studies show that lack of SP-R210 isoforms does not impair IAV binding and internalization. Lack of SP-R210L, however, results in macropinocytic retention of the virus that depends on both SP-R210S and interferon-inducible transmembrane protein-3 (IFITM3). Mass spectrometry and Western blot analyses indicate that SP-R210 isoforms modulate differential recruitment of the Rho-family GTPase RAC1 and guanine nucleotide exchange factors. Our study suggests that SP-R210 isoforms modulate RAC-dependent macropinosomal sorting of IAV to discrete endosomal and lysosomal compartments that either permit or prevent endolysosomal escape and inflammatory sensing of viral genomes in macrophages.

Game of Omes: ribosome profiling expands the MHC-I immunopeptidome.

Peptide ligands presented by cell-surface MHC class-I molecules enable T cells to eradicate intracellular pathogens and cancers. The presented peptide repertoire, the class-I immunopeptidome, is generated from each cell's translatome in a highly biased manner to avoid overrepresenting highly abundant translation products. The immunopeptidome can only be defined by mass spectrometry (MS). Here, we review recent advances in immunopeptidomics, focusing on using ribosome profiling as the optimal MS database to optimize the false- and failed-discovery rates and relate these findings to the contribution of defective ribosomal products and cellular quality control mechanisms to MHC class-I antigen processing and presentation.

Surfactant protein A alters endosomal trafficking of influenza A virus in macrophages.

Influenza A virus infection (IAV) often leads to acute lung injury that impairs breathing and can lead to death, with disproportionate mortality in children and the elderly. Surfactant Protein A (SP-A) is a calcium-dependent opsonin that binds a variety of pathogens to help control pulmonary infections by alveolar macrophages. Alveolar macrophages play critical roles in host resistance and susceptibility to IAV infection. The effect of SP-A on IAV infection and antiviral response of macrophages, however, is not understood. Here, we report that SP-A attenuates IAV infection in a dose-dependent manner at the level of endosomal trafficking, resulting in infection delay in a model macrophage cell line. The ability of SP-A to suppress infection was independent of its glycosylation status. Binding of SP-A to hemagglutinin did not rely on the glycosylation status or sugar binding properties of either protein. Incubation of either macrophages or IAV with SP-A slowed endocytic uptake rate of IAV. SP-A interfered with binding to cell membrane and endosomal exit of the viral genome as indicated by experiments using isolated cell membranes, an antibody recognizing a pH-sensitive conformational epitope on hemagglutinin, and microscopy. Lack of SP-A in mice enhanced IFNß expression, viral clearance and reduced mortality from IAV infection. These findings support the idea that IAV is an opportunistic pathogen that co-opts SP-A to evade host defense by alveolar macrophages. Our study highlights novel aspects of host-pathogen interactions that may lead to better understanding of the local mechanisms that shape activation of antiviral and inflammatory responses to viral infection in the lung.

Rapalogs downmodulate intrinsic immunity and promote cell entry of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in immunocompromised individuals is associated with prolonged virus shedding and evolution of viral variants. Rapamycin and its analogs (rapalogs, including everolimus, temsirolimus, and ridaforolimus) are FDA approved as mTOR inhibitors for the treatment of human diseases, including cancer and autoimmunity. Rapalog use is commonly associated with an increased susceptibility to infection, which has been traditionally explained by impaired adaptive immunity. Here, we show that exposure to rapalogs increased susceptibility to SARS-CoV-2 infection in tissue culture and in immunologically naive rodents by antagonizing the cell-intrinsic immune response. We identified 1 rapalog (ridaforolimus) that was less potent in this regard and demonstrated that rapalogs promote spike-mediated entry into cells, by triggering the degradation of the antiviral proteins IFITM2 and IFITM3 via an endolysosomal remodeling program called microautophagy. Rapalogs that increased virus entry inhibited mTOR-mediated phosphorylation of the transcription factor TFEB, which facilitated its nuclear translocation and triggered microautophagy. In rodent models of infection, injection of rapamycin prior to and after virus exposure resulted in elevated SARS-CoV-2 replication and exacerbated viral disease, while ridaforolimus had milder effects. Overall, our findings indicate that preexisting use of certain rapalogs may elevate host susceptibility to SARS-CoV-2 infection and disease by activating lysosome-mediated suppression of intrinsic immunity.

Broadly neutralizing antibodies target the coronavirus fusion peptide.

The potential for future coronavirus outbreaks highlights the need to broadly target this group of pathogens. We used an epitope-agnostic approach to identify six monoclonal antibodies that bind to spike proteins from all seven human-infecting coronaviruses. All six antibodies target the conserved fusion peptide region adjacent to the S2' cleavage site. COV44-62 and COV44-79 broadly neutralize alpha- and betacoronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants BA.2 and BA.4/5, albeit with lower potency than receptor binding domain-specific antibodies. In crystal structures of COV44-62 and COV44-79 antigen-binding fragments with the SARS-CoV-2 fusion peptide, the fusion peptide epitope adopts a helical structure and includes the arginine residue at the S2' cleavage site. COV44-79 limited disease caused by SARS-CoV-2 in a Syrian hamster model. These findings highlight the fusion peptide as a candidate epitope for next-generation coronavirus vaccine development.

MHC Class I Immunopeptidome: Past, Present, and Future.

In the 35 years since the revelation that short peptides bound to major histocompatibility complex class I and II molecules are the secret of the major histocompatibility complex-restricted nature of T-cell recognition, there has been enormous progress in characterizing the immunopeptidome, the repertoire of peptide presented for immunosurveillance. Here, the major milestones in the journey are marked, the contribution of proteasome-mediated splicing to the immunopeptidome is discussed, and exciting recent findings relating the immunopeptidome to the translatome revealed by ribosome profiling (RiboSeq) is detailed. Finally, what is needed for continued progress is opined about, which includes the infusion of talented young scientists into the antigen-processing field, currently undergoing a renaissance; thanks in part to the astounding success of T-cell-based cancer immunotherapy.

Rapalogs downmodulate intrinsic immunity and promote cell entry of SARS-CoV-2.

SARS-CoV-2 infection in immunocompromised individuals is associated with prolonged virus shedding and evolution of viral variants. Rapamycin and its analogs (rapalogs, including everolimus, temsirolimus, and ridaforolimus) are FDA-approved as mTOR inhibitors for the treatment of human diseases, including cancer and autoimmunity. Rapalog use is commonly associated with increased susceptibility to infection, which has been traditionally explained by impaired adaptive immunity. Here, we show that exposure to rapalogs increases susceptibility to SARS-CoV-2 infection in tissue culture and in immunologically naive rodents by antagonizing the cell-intrinsic immune response. By identifying one rapalog (ridaforolimus) that is less potent in this regard, we demonstrate that rapalogs promote Spike-mediated entry into cells by triggering the degradation of antiviral proteins IFITM2 and IFITM3 via an endolysosomal remodeling program called microautophagy. Rapalogs that increase virus entry inhibit the mTOR-mediated phosphorylation of the transcription factor TFEB, which facilitates its nuclear translocation and triggers microautophagy. In rodent models of infection, injection of rapamycin prior to and after virus exposure resulted in elevated SARS-CoV-2 replication and exacerbated viral disease, while ridaforolimus had milder effects. Overall, our findings indicate that preexisting use of certain rapalogs may elevate host susceptibility to SARS-CoV-2 infection and disease by activating lysosome-mediated suppression of intrinsic immunity.

Generation and characterization of HLA-A2 transgenic mice expressing the human TCR 1G4 specific for the HLA-A2 restricted NY-ESO-1157-165 tumor-specific peptide.

BACKGROUND: NY-ESO-1 is a tumor-specific, highly immunogenic, human germ cell antigen of the MAGE-1 family that is a promising vaccine and cell therapy candidate in clinical trial development. The mouse genome does not encode an NY-ESO-1 homolog thereby not subjecting transgenic T-cells to thymic tolerance mechanisms that might impair in-vivo studies. We hypothesized that an NY-ESO-1 T cell receptor (TCR) transgenic mouse would provide the unique opportunity to study avidity of TCR response against NY-ESO-1 for tumor vaccine and cellular therapy development against this clinically relevant and physiological human antigen. METHODS: To study in vitro and in vivo the requirements for shaping an effective T cell response against the clinically relevant NY-ESO-1, we generated a C57BL/6 HLA-A*0201 background TCR transgenic mouse encoding the 1G4 TCR specific for the human HLA-A2 restricted, NY-ESO-1157-165 SLLMWITQC (9C), initially identified in an NY-ESO-1 positive melanoma patient. RESULTS: The HLA-A*0201 restricted TCR was positively selected on both CD4+ and CD8+ cells. Mouse 1G4 T cells were not activated by endogenous autoimmune targets or a large library of non-cognate viral antigens. In contrast, their activation by HLA-A2 NY-ESO-1157-165 complexes was evident by proliferation, CD69 upregulation, interferon-γ production, and interleukin-2 production, and could be tuned using a twofold higher affinity altered peptide ligand, NY-ESO-1157-165V. NY-ESO-1157-165V recombinant vaccination of syngeneic mice adoptively transferred with m1G4 CD8+ T cells controlled tumor growth in vivo. 1G4 transgenic mice suppressed growth of syngeneic methylcholanthrene (MCA) induced HHD tumor cells expressing the full-length human NY-ESO-1 protein but not MCA HHD tumor cells lacking NY-ESO-1. CONCLUSIONS: The 1G4 TCR mouse model for the physiological human TCR against the clinically relevant antigen, NY-ESO-1, is a valuable tool with the potential to accelerate clinical development of NY-ESO-1-targeted T-cell and vaccine therapies.

Cutting Edge: Myosin 18A Is a Novel Checkpoint Regulator in B Cell Differentiation and Antibody-Mediated Immunity.

We investigated the function of the newly discovered myosin family protein myosin 18A (Myo18A) in Ab-mediated immunity by generating B cell-conditional Myo18A-deficient mice. Myo18A deficiency led to expansion of bone marrow progenitor B cells and mature B cells in secondary lymphoid organs. Myo18A-deficient mice displayed serum IgM hyperglobulinemia and increased splenic IgM-secreting cells, with older mice switching to IgG1 hyperglobulinemia and autoantibody development. Immunization of Myo18A-deficient mice with inactivated influenza virus led to development of more potent neutralizing Abs against the major Ag hemagglutinin, associated with persistent accumulation of Ag-specific germinal center B cells and more Ag-specific bone marrow plasma cells. In vitro stimulation with TLR7 and BCR ligands revealed a greater ability of Myo18A-deficient B cells to differentiate into Ab-secreting cells, associated with higher AID and Blimp-1 expression. Overall, our study demonstrates that Myo18A is a novel negative regulator of B cell homeostasis, differentiation, and humoral immunity.

Most non-canonical proteins uniquely populate the proteome or immunopeptidome.

Combining RNA sequencing, ribosome profiling, and mass spectrometry, we elucidate the contribution of non-canonical translation to the proteome and major histocompatibility complex (MHC) class I immunopeptidome. Remarkably, of 14,498 proteins identified in three human B cell lymphomas, 2,503 are non-canonical proteins. Of these, 28% are novel isoforms and 72% are cryptic proteins encoded by ostensibly non-coding regions (60%) or frameshifted canonical genes (12%). Cryptic proteins are translated as efficiently as canonical proteins, have more predicted disordered residues and lower stability, and critically generate MHC-I peptides 5-fold more efficiently per translation event. Translating 5' "untranslated" regions hinders downstream translation of genes involved in transcription, translation, and antiviral responses. Novel protein isoforms show strong enrichment for signaling pathways deregulated in cancer. Only a small fraction of cryptic proteins detected in the proteome contribute to the MHC-I immunopeptidome, demonstrating the high preferential access of cryptic defective ribosomal products to the class I pathway.

Immune MAL2-practice: breast cancer immunoevasion via MHC class I degradation.

The success of tumor immunotherapy, while partial, confirms the existence and importance of tumor immunosurveillance. CD8+ T cell recognition of tumor-specific peptides bound to MHC class I (MHC-I) molecules is central to this process. In this issue of the JCI, Fang, Wang, et al. describe a unique tumor immunoevasion strategy based on endocytosis and degradation of MHC-I complexes mediated by the trafficking factor MAL2. Notably, MAL2 expression was associated with poor prognosis of breast cancer, and its downregulation enhanced CD8+ T cell recognition of breast cancer in various experimental models. This work demonstrates that a deeper understanding of tumor interference with MHC-I stability and trafficking has considerable potential for enhancing immunotherapies.

Genome-wide Screens Identify Lineage- and Tumor-Specific Genes Modulating MHC-I- and MHC-II-Restricted Immunosurveillance of Human Lymphomas.

Tumors frequently subvert major histocompatibility complex class I (MHC-I) peptide presentation to evade CD8+ T cell immunosurveillance, though how this is accomplished is not always well defined. To identify the global regulatory networks controlling antigen presentation, we employed genome-wide screening in human diffuse large B cell lymphomas (DLBCLs). This approach revealed dozens of genes that positively and negatively modulate MHC-I cell surface expression. Validated genes clustered in multiple pathways including cytokine signaling, mRNA processing, endosomal trafficking, and protein metabolism. Genes can exhibit lymphoma subtype- or tumor-specific MHC-I regulation, and a majority of primary DLBCL tumors displayed genetic alterations in multiple regulators. We established SUGT1 as a major positive regulator of both MHC-I and MHC-II cell surface expression. Further, pharmacological inhibition of two negative regulators of antigen presentation, EZH2 and thymidylate synthase, enhanced DLBCL MHC-I presentation. These and other genes represent potential targets for manipulating MHC-I immunosurveillance in cancers, infectious diseases, and autoimmunity.

A few good peptides: MHC class I-based cancer immunosurveillance and immunoevasion.

The remarkable success of immune checkpoint inhibitors demonstrates the potential of tumour-specific CD8+ T cells to prevent and treat cancer. Although the number of lives saved by immunotherapy mounts, only a relatively small fraction of patients are cured. Here, we review two of the factors that limit the application of CD8+ T cell immunotherapies: difficulties in identifying tumour-specific peptides presented by MHC class I molecules and the ability of tumour cells to impair antigen presentation as they evolve under T cell selection. We describe recent advances in understanding how peptides are generated from non-canonical translation of defective ribosomal products, relate this to the dysregulated translation that is a feature of carcinogenesis and propose dysregulated translation as an important new source of tumour-specific peptides. We discuss how the synthesis and function of components of the antigen-processing and presentation pathway, including the recently described immunoribosome, are manipulated by tumours for immunoevasion and point to common druggable targets that may enhance immunotherapy.

A Hyper-IgM Syndrome Mutation in Activation-Induced Cytidine Deaminase Disrupts G-Quadruplex Binding and Genome-wide Chromatin Localization.

Precise targeting of activation-induced cytidine deaminase (AID) to immunoglobulin (Ig) loci promotes antibody class switch recombination (CSR) and somatic hypermutation (SHM), whereas AID targeting of non-Ig loci can generate oncogenic DNA lesions. Here, we examined the contribution of G-quadruplex (G4) nucleic acid structures to AID targeting in vivo. Mice bearing a mutation in Aicda (AIDG133V) that disrupts AID-G4 binding modeled the pathology of hyper-IgM syndrome patients with an orthologous mutation, lacked CSR and SHM, and had broad defects in genome-wide AIDG133V chromatin localization. Genome-wide analyses also revealed that wild-type AID localized to MHCII genes, and AID expression correlated with decreased MHCII expression in germinal center B cells and diffuse large B cell lymphoma. Our findings indicate a crucial role for G4 binding in AID targeting and suggest that AID activity may extend beyond Ig loci to regulate the expression of genes relevant to the physiology and pathology of activated B cells.

Author Correction: A few good peptides: MHC class I-based cancer immunosurveillance and immunoevasion.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

DRiPs get molecular.

The defective ribosomal product (DRiP) hypothesis was proposed nearly 25 years ago to account for the rapid generation of peptides from otherwise metabolically stable viral proteins. It posits that errors in converting genetic information into stable proteins accounts for a sizeable fraction of the immunopeptidome. Here, we review recent studies that provide insight into the importance of DRiPs for immunosurveillance and the myriad mechanisms that give rise to DRiPs.

Myc controls a distinct transcriptional program in fetal thymic epithelial cells that determines thymus growth.

Interactions between thymic epithelial cells (TEC) and developing thymocytes are essential for T cell development, but molecular insights on TEC and thymus homeostasis are still lacking. Here we identify distinct transcriptional programs of TEC that account for their age-specific properties, including proliferation rates, engraftability and function. Further analyses identify Myc as a regulator of fetal thymus development to support the rapid increase of thymus size during fetal life. Enforced Myc expression in TEC induces the prolonged maintenance of a fetal-specific transcriptional program, which in turn extends the growth phase of the thymus and enhances thymic output; meanwhile, inducible expression of Myc in adult TEC similarly promotes thymic growth. Mechanistically, this Myc function is associated with enhanced ribosomal biogenesis in TEC. Our study thus identifies age-specific transcriptional programs in TEC, and establishes that Myc controls thymus size.

Hemagglutinin head-specific responses dominate over stem-specific responses following prime boost with mismatched vaccines.

Broadly neutralizing Abs targeting the HA stem can provide broad protection against different influenza subtypes, raising the question of how best to elicit such Abs. We have previously demonstrated that vaccination with pandemic live-attenuated influenza vaccine (pLAIV) establishes immune memory for HA head-specific Abs. Here, we determine the extent to which matched versus mismatched LAIV-inactivated subunit vaccine (IIV) prime-boost vaccination elicits stem-specific memory B cells and Abs. We vaccinated African green monkeys with H5N1 pLAIV-pIIV or H5N1 pLAIV followed by seasonal IIV (sIIV) or with H5N1 pLAIV alone and measured Abs and HA-specific B cell responses. While we observed an increase in stem-specific memory B cells, head-specific memory B cell responses were substantially higher than stem-specific responses and were dominant even following boost with mismatched IIV. Neutralizing Abs against heterologous influenza viruses were undetectable. Head-specific B cells from draining lymph nodes exhibited germinal center markers, while stem-specific B cells found in the spleen and peripheral blood did not. Thus, although mismatched prime-boost generated a pool of stem-specific memory B cells, head-specific B cells and serum Abs substantially dominated the immune response. These findings have implications for including full-length native HA in prime-boost strategies intended to induce stem-specific Abs for universal influenza vaccination.

Peptide Channeling: The Key to MHC Class I Immunosurveillance?

MHC class I presentation of short peptides enables CD8+ T cell (TCD8+) immunosurveillance of tumors and intracellular pathogens. A key feature of the class I pathway is that the immunopeptidome is highly skewed from the cellular degradome, indicating high selectivity of the access of protease-generated peptides to class I molecules. Similarly, in professional antigen-presenting cells, peptides from minute amounts of proteins introduced into the cytosol outcompete an overwhelming supply of constitutively generated peptides. Here, we propose that antigen processing is based on substrate channeling and review recent studies from the antigen processing and cell biology fields that provide a starting point for testing this hypothesis.