The show and tell of cross-presentation.

Cross-presentation is the culmination of complex subcellular processes that allow the processing of exogenous proteins and the presentation of resultant peptides on major histocompatibility class I (MHC-I) molecules to CD8 T cells. Dendritic cells (DCs) are a cell type that uniquely specializes in cross-presentation, mainly in the context of viral or non-viral infection and cancer. DCs have an extensive network of endovesicular pathways that orchestrate the biogenesis of an ideal cross-presentation compartment where processed antigen, MHC-I molecules, and the MHC-I peptide loading machinery all meet. As a central conveyor of information to CD8 T cells, cross-presentation allows cross-priming of T cells which carry out robust adaptive immune responses for tumor and viral clearance. Cross-presentation can be canonical or noncanonical depending on the functional status of the transporter associated with antigen processing (TAP), which in turn influences the vesicular route of MHC-I delivery to internalized antigen and the cross-presented repertoire of peptides. Because TAP is a central node in MHC-I presentation, it is targeted by immune evasive viruses and cancers. Thus, understanding the differences between canonical and noncanonical cross-presentation may inform new therapeutic avenues against cancer and infectious disease. Defects in cross-presentation on a cellular and genetic level lead to immune-related disease progression, recurrent infection, and cancer progression. In this chapter, we review the process of cross-presentation beginning with the DC subsets that conduct cross-presentation, the signals that regulate cross-presentation, the vesicular trafficking pathways that orchestrate cross-presentation, the modes of cross-presentation, and ending with disease contexts where cross-presentation plays a role.

TAP-ing into the cross-presentation secrets of dendritic cells.

Viral blockade of the transporter associated with antigen processing (TAP) diminishes surface and endosomal recycling compartment levels of major histocompatibility complex class-I (MHC-I) in dendritic cells (DCs), and compromises both classical MHC-I presentation and canonical cross-presentation during infection to impair CD8 T-cell immunity. Virus-specific CD8 T cells are thought to be cross-primed mostly by uninfected TAP-sufficient DCs through cross-presentation of viral peptides from internalized virus-infected dying cells. The dilemma is that CD8 T cells primed to TAP-dependent viral peptides are mismatched to the TAP-independent epitopes presented on tissues infected with immune-evasive viruses. Noncanonical cross-presentation in DCs overcomes cell-intrinsic TAP blockade to nevertheless prime protective TAP-independent CD8 T cells best-matched against the infection. Exploitation of noncanonical cross-presentation may prevent chronic infections with immune-evasive viruses. It may also control immune-evasive cancers that have downmodulated TAP expression.

Apoptotic cell death in disease-Current understanding of the NCCD 2023.

Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease.

Different routes of MHC-I delivery to phagosomes and their consequences to CD8 T cell immunity.

Dendritic cells (DCs) present internalized antigens to CD8 T cells through cross-presentation by major histocompatibility complex class I (MHC-I) molecules. While conventional cDC1 excel at cross-presentation, cDC2 can be licensed to cross-present during infection by signals from inflammatory receptors, most prominently Toll-like receptors (TLRs). At the core of the regulation of cross-presentation by TLRs is the control of subcellular MHC-I traffic. Within DCs, MHC-I are enriched within endosomal recycling compartments (ERC) and traffic to microbe-carrying phagosomes under the control of phagosome-compartmentalized TLR signals to favor CD8 T cell cross-priming to microbial antigens. Viral blockade of the transporter associated with antigen processing (TAP), known to inhibit the classic MHC-I presentation of cytoplasmic protein-derived peptides, depletes the ERC stores of MHC-I to simultaneously also block TLR-regulated cross-presentation. DCs counter this impairment in the two major pathways of MHC-I presentation to CD8 T cells by mobilizing noncanonical cross-presentation, which delivers MHC-I to phagosomes from a new location in the ER-Golgi intermediate compartment (ERGIC) where MHC-I abnormally accumulate upon TAP blockade. Noncanonical cross-presentation thus rescues MHC-I presentation and cross-primes TAP-independent CD8 T cells best-matched against target cells infected with immune evasive viruses. Because noncanonical cross-presentation relies on a phagosome delivery route of MHC-I that is not under TLR control, it risks potential cross-presentation of self-antigens during infection. Here I review these findings to illustrate how the subcellular route of MHC-I to phagosomes critically impacts the regulation of cross-presentation and the nature of the CD8 T cell response to infection and cancer. I highlight important and novel implications to CD8 T cell vaccines and immunotherapy.

Network analysis of large-scale ImmGen and Tabula Muris datasets highlights metabolic diversity of tissue mononuclear phagocytes.

The diversity of mononuclear phagocyte (MNP) subpopulations across tissues is one of the key physiological characteristics of the immune system. Here, we focus on understanding the metabolic variability of MNPs through metabolic network analysis applied to three large-scale transcriptional datasets: we introduce (1) an ImmGen MNP open-source dataset of 337 samples across 26 tissues; (2) a myeloid subset of ImmGen Phase I dataset (202 MNP samples); and (3) a myeloid mouse single-cell RNA sequencing (scRNA-seq) dataset (51,364 cells) assembled based on Tabula Muris Senis. To analyze such large-scale datasets, we develop a network-based computational approach, genes and metabolites (GAM) clustering, for unbiased identification of the key metabolic subnetworks based on transcriptional profiles. We define 9 metabolic subnetworks that encapsulate the metabolic differences within MNP from 38 different tissues. Obtained modules reveal that cholesterol synthesis appears particularly active within the migratory dendritic cells, while glutathione synthesis is essential for cysteinyl leukotriene production by peritoneal and lung macrophages.

Caspase-11 interaction with NLRP3 potentiates the noncanonical activation of the NLRP3 inflammasome.

Caspase-11 detection of intracellular lipopolysaccharide (LPS) from invasive Gram-negative bacteria mediates noncanonical activation of the NLRP3 inflammasome. While avirulent bacteria do not invade the cytosol, their presence in tissues necessitates clearance and immune system mobilization. Despite sharing LPS, only live avirulent Gram-negative bacteria activate the NLRP3 inflammasome. Here, we found that bacterial mRNA, which signals bacterial viability, was required alongside LPS for noncanonical activation of the NLRP3 inflammasome in macrophages. Concurrent detection of bacterial RNA by NLRP3 and binding of LPS by pro-caspase-11 mediated a pro-caspase-11-NLRP3 interaction before caspase-11 activation and inflammasome assembly. LPS binding to pro-caspase-11 augmented bacterial mRNA-dependent assembly of the NLRP3 inflammasome, while bacterial viability and an assembled NLRP3 inflammasome were necessary for activation of LPS-bound pro-caspase-11. Thus, the pro-caspase-11-NLRP3 interaction nucleated a scaffold for their interdependent activation explaining their functional reciprocal exclusivity. Our findings inform new vaccine adjuvant combinations and sepsis therapy.

Spotlight on TAP and its vital role in antigen presentation and cross-presentation.

In the late 1980s and early 1990s, the hunt for a transporter molecule ostensibly responsible for the translocation of peptides across the endoplasmic reticulum (ER) membrane yielded the successful discovery of transporter associated with antigen processing (TAP) protein. TAP is a heterodimer complex comprised of TAP1 and TAP2, which utilizes ATP to transport cytosolic peptides into the ER across its membrane. In the ER, together with other components it forms the peptide loading complex (PLC), which directs loading of high affinity peptides onto nascent major histocompatibility complex class I (MHC-I) molecules that are then transported to the cell surface for presentation to CD8+ T cells. TAP also plays a crucial role in transporting peptides into phagosomes and endosomes during cross-presentation in dendritic cells (DCs). Because of the critical role that TAP plays in both classical MHC-I presentation and cross-presentation, its expression and function are often compromised by numerous types of cancers and viruses to evade recognition by cytotoxic CD8 T cells. Here we review the discovery and function of TAP with a major focus on its role in cross-presentation in DCs. We discuss a recently described emergency route of noncanonical cross-presentation that is mobilized in DCs upon TAP blockade to restore CD8 T cell cross-priming. We also discuss the various strategies employed by cancer cells and viruses to target TAP expression or function to evade immunosurveillance - along with some strategies by which the repertoire of peptides presented by cells which downregulate TAP can be targeted as a therapeutic strategy to mobilize a TAP-independent CD8 T cell response. Lastly, we discuss TAP polymorphisms and the role of TAP in inherited disorders.

TAP dysfunction in dendritic cells enables noncanonical cross-presentation for T cell priming.

Classic major histocompatibility complex class I (MHC-I) presentation relies on shuttling cytosolic peptides into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). Viruses disable TAP to block MHC-I presentation and evade cytotoxic CD8+ T cells. Priming CD8+ T cells against these viruses is thought to rely solely on cross-presentation by uninfected TAP-functional dendritic cells. We found that protective CD8+ T cells could be mobilized during viral infection even when TAP was absent in all hematopoietic cells. TAP blockade depleted the endosomal recycling compartment of MHC-I molecules and, as such, impaired Toll-like receptor-regulated cross-presentation. Instead, MHC-I molecules accumulated in the ER-Golgi intermediate compartment (ERGIC), sequestered away from Toll-like receptor control, and coopted ER-SNARE Sec22b-mediated vesicular traffic to intersect with internalized antigen and rescue cross-presentation. Thus, when classic MHC-I presentation and endosomal recycling compartment-dependent cross-presentation are impaired in dendritic cells, cell-autonomous noncanonical cross-presentation relying on ERGIC-derived MHC-I counters TAP dysfunction to nevertheless mediate CD8+ T cell priming.

Increasing complexity of NLRP3 inflammasome regulation.

Inflammasomes are multiprotein complexes that assemble upon detection of danger signals to activate the inflammatory enzyme caspase-1, trigger secretion of the highly proinflammatory cytokine IL-1ß, and induce an inflammatory cell death called pyroptosis. Distinctiveness of the nucleotide-binding oligomerization (NOD), Leucine-rich repeat (LRR)-containing protein (NLRP3) inflammasome resides in the diversity of molecules that induce its activation, indicating a certain intricacy. Furthermore, besides the canonical activation of NLRP3 in response to various stimuli, caspase-11-dependent detection of intracellular LPS activates NLRP3 through a noncanonical pathway. Several aspects of the NLRP3 inflammasome are not characterized or remain unclear. In this review, we summarize the different modes of NLRP3 activation. We describe recent insights into post-translational and cellular regulation that confer further complexity to NLRP3 inflammasomes.

A Comprehensive Experimental Guide to Studying Cross-Presentation in Dendritic Cells In Vitro.

Cross-presentation was first observed serendipitously in the 1970s. The importance of it was quickly realized and subsequently attracted great attention from immunologists. Since then, our knowledge of the ability of certain antigen presenting cells to internalize, process, and load exogenous antigens onto MHC-I molecules to cross-prime CD8+ T cells has increased significantly. Dendritic cells (DCs) are exceptional cross-presenters, thus making them a great tool to study cross-presentation but the relative rarity of DCs in circulation and in tissues makes it challenging to isolate sufficient numbers of cells to study this process in vitro. In this paper, we describe in detail two methods to culture DCs from bone-marrow progenitors and a method to expand the numbers of DCs present in vivo as a source of endogenous bona-fide cross-presenting DCs. We also describe methods to assess cross-presentation by DCs using the activation of primary CD8+ T cells as a readout. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Isolation of bone marrow progenitor cells Basic Protocol 2: In vitro differentiation of dendritic cells with GM-CSF Support Protocol 1: Preparation of conditioned medium from GM-CSF producing J558L cells Basic Protocol 3: In vitro differentiation of dendritic cells with Flt3L Support Protocol 2: Preparation of Flt3L containing medium from B16-Flt3L cells Basic Protocol 4: Expansion of cDC1s in vivo for use in ex vivo experiments Basic Protocol 5: Characterizing resting and activated dendritic cells Basic Protocol 6: Dendritic cell stimulation, antigenic cargo, and fixation Support Protocol 3: Preparation of model antigen coated microbeads Support Protocol 4: Preparation of apoptotic cells Support Protocol 5: Preparation of recombinant bacteria Basic Protocol 7: Immunocytochemistry immunofluorescence (ICC/IF) Support Protocol 6: Preparation of Alcian blue-coated coverslips Basic Protocol 8: CD8+ T cell activation to assess cross-presentation Support Protocol 7: Isolation and labeling of CD8+ T cells with CFSE.

Macrophages Maintain Epithelium Integrity by Limiting Fungal Product Absorption.

The colon is primarily responsible for absorbing fluids. It contains a large number of microorganisms including fungi, which are enriched in its distal segment. The colonic mucosa must therefore tightly regulate fluid influx to control absorption of fungal metabolites, which can be toxic to epithelial cells and lead to barrier dysfunction. How this is achieved remains unknown. Here, we describe a mechanism by which the innate immune system allows rapid quality check of absorbed fluids to avoid intoxication of colonocytes. This mechanism relies on a population of distal colon macrophages that are equipped with "balloon-like" protrusions (BLPs) inserted in the epithelium, which sample absorbed fluids. In the absence of macrophages or BLPs, epithelial cells keep absorbing fluids containing fungal products, leading to their death and subsequent loss of epithelial barrier integrity. These results reveal an unexpected and essential role of macrophages in the maintenance of colon-microbiota interactions in homeostasis. VIDEO ABSTRACT.

Caspase-8-Dependent Inflammatory Responses Are Controlled by Its Adaptor, FADD, and Necroptosis.

Cell death pathways regulate various homeostatic processes. Autoimmune lymphoproliferative syndrome (ALPS) in humans and lymphoproliferative (LPR) disease in mice result from abrogated CD95-induced apoptosis. Because caspase-8 mediates CD95 signaling, we applied genetic approaches to dissect the roles of caspase-8 in cell death and inflammation. Here, we describe oligomerization-deficient Caspase-8F122GL123G/F122GL123G and non-cleavable Caspase-8D387A/D387A mutant mice with defective caspase-8-mediated apoptosis. Although neither mouse developed LPR disease, removal of the necroptosis effector Mlkl from Caspase-8D387A/D387A mice revealed an inflammatory role of caspase-8. Ablation of one allele of Fasl, Fadd, or Ripk1 prevented the pathology of Casp8D387A/D387AMlkl-/- animals. Removing both Fadd alleles from these mice resulted in early lethality prior to post-natal day 15 (P15), which was prevented by co-ablation of either Ripk1 or Caspase-1. Our results suggest an in vivo role of the inflammatory RIPK1-caspase-8-FADD (FADDosome) complex and reveal a FADD-independent inflammatory role of caspase-8 that involves activation of an inflammasome.

MerTK Blockade Fuels Anti-tumor Immunity.

Phagocytosis of apoptotic cells via the receptor MerTK is important for immune tolerance. In this issue of Immunity, Zhou et al. report that blockade of MerTK-mediated phagocytosis mobilizes anti-tumor immunity through a mechanism that involves the transport of tumor-derived cGAMP into macrophages via the ATP-activated channel P2X7R.

On cell death in the intestinal epithelium and its impact on gut homeostasis.

PURPOSE OF REVIEW: Both apoptotic and nonapoptotic cell extrusion preserve the barrier functions of epithelia. Live cell extrusion is the paradigm for homeostatic renewal of intestinal epithelial cells (IEC). By extension, as extruded cells are not apoptotic, this form of cell shedding is thought to be largely ignored by lamina propria phagocytes and without immune consequence. RECENT FINDINGS: Visualization of apoptotic IEC inside distinct subsets of intestinal phagocytes during homeostasis has highlighted apoptosis as a normal component of the natural turnover of the intestinal epithelium. Analysis of phagocytes with or without apoptotic IEC corpses has shown how apoptotic IEC constrain inflammatory pathways within phagocytes and induce immunosuppressive regulatory CD4 T-cell differentiation. Many of the genes involved overlap with susceptibility genes for inflammatory bowel disease (IBD). SUMMARY: Excessive IEC death and loss-of-barrier function is characteristic of IBD. As regulatory and tolerogenic mechanisms are broken in IBD, a molecular understanding of the precise triggers and modes of IEC death as well as their consequences on intestinal inflammation is necessary. This characterization should guide new therapies that restore homeostatic apoptosis, along with its associated programs of immune tolerance and immunosuppression, to achieve mucosal healing and long-term remission.

Exploiting vita-PAMPs in vaccines.

Live attenuated vaccines elicit stronger protective immunity than dead vaccines. Distinct PAMPs designated as vita-PAMPs signify microbial viability to innate immune cells. Two vita-PAMPs have been characterized: cyclic-di-adenosine-monophosphate (c-di-AMP) and prokaryotic messenger RNA (mRNA). c-di-AMP produced by live Gram-positive bacteria elicits augmented production of STING-dependent type-I interferon, whereas prokaryotic mRNA from live bacteria is detected by TLR8 enabling discrimination of live from dead bacteria. Bacterial mRNA from live Gram-negative bacteria triggers a heightened type-I interferon and NLRP3 inflammasome response. By mobilizing unique viability-associated innate responses, vita-PAMPs mobilize adaptive immunity that best elicits protection, including follicular T helper cell and antibody responses. Here, we review the molecular mechanisms that confer the unique adjuvanticity of vita-PAMPs and discuss their applications in vaccine design.

Detection of a vita-PAMP STINGs cells into reticulophagy.

Phagocytes cope with the threat of living bacteria via detection of vita-PAMPs, a specific class of pathogen-associated molecular patterns (PAMPs) that denotes microbial viability and trigger a commensurate innate response. Prokaryotic mRNA and cyclic-di-adenosine monophosphate (c-di-AMP) serve as vita-PAMPs for Gram-negative and Gram-positive bacteria, respectively, and elicit heightened proinflammatory responses not warranted for dead bacteria. The innate sensor TMEM173/STING detects c-di-AMP produced by internalized live Gram-positive bacteria, and quickly mobilizes interdependent pre-formed cell-autonomous responses including endoplasmic reticulum (ER) stress, MTOR inactivation, and reticulophagy. In turn, reticulophagy serves a dual role in restoring phagocyte homeostasis and orchestrating a type I IFN response. ER-stress induced macroautophagy/autophagy sequesters stressed ER, resolves ER stress and prevents apoptosis in response to live bacteria. Reticulophagy relocalizes ER-resident TMEM173/STING to phagophores, which then act as TMEM173/STING-signaling compartments. Here, we discuss our findings in the context of innate immunity and cell homeostasis.