Critical illness, including COVID-19 and trauma, induce immune dysregulation associated with morbidity and mortality. Direct comparison is crucial to identify shared pathobiological mechanisms to guide precision therapies. This prospective cohort study undertook a comprehensive, longitudinal comparison of innate immune cell function and plasma protein expression in critically ill patients at days 1, 3, and 5 of intensive care unit admission. We enrolled 26 COVID-19 patients, 20 trauma patients, and 18 healthy controls and analysed plasma proteins and immune cell phenotypes and responses to S. aureus bioparticles. An unsupervised multi-omics factor analysis (MOFA) identified shared sources of biological variation. Here we show shared, profound immune alterations, including neutrophilia, decreased non-classical monocytes and dendritic cells (DCs), and a hyper-phagocytic and reactive oxygen species producing neutrophil state in both patient cohorts compared to healthy controls. Classical monocytes from patients were unable to upregulate CD11c, CD86, and SIRPα upon stimulation, and a phagocytosis-impaired SIRPα-CD11b- DC population was enriched from day 1. Innate immune activation profiles could segregate patients and were associated with subsequent impaired innate/adaptive crosstalk. This direct comparison of severe COVID-19 and trauma patients demonstrates many shared immune alterations between these distinct critical illnesses, which may benefit from similar targeted immunomodulation.

The development of unconventional αβ T cells, including invariant natural killer T (iNKT) and mucosal-associated invariant T (MAIT) cells, in the thymus is distinct from conventional T cells. Unconventional αβ T cells adopt a memory phenotype, acquire effector functions, and can reside in the thymus long-term. It is well-established that positive selection of these unconventional T cells from CD4+CD8+ double-positive (DP) precursors depends on interaction with other DP cells. However, postselection, the regulation of their maturation and effector differentiation is less well understood. Professional antigen-presenting cells (APCs) are thought to have a role, but their roles have only been partially investigated previously. In this study, we investigate the impact of perturbing thymic dendritic cell (DC) and macrophage populations on intrathymic iNKT and MAIT effector subsets in C57BL/6 mice. We show that conventional type 1 DCs (cDC1s) support iNKT1 cells, while CX3CR1- and Mgl2-expressing cDC2s and macrophages support MAIT17 cells. Lastly, we show that disrupting the XCR1-XCL1 axis, which was previously shown to control cDC1 localization to the thymic cortex, alters the balance between iNKT and MAIT cells in the thymus, where there is augmentation of the iNKT cell compartment at the expense of MAIT cells. These findings further highlight the roles of hematopoietic APCs in supporting intrathymic unconventional αβ T cells.

Abstract Background Acute lung injury (ALI) after extracorporeal circulation can originate from multiple causes, including transfusion. Despite improvements in blood safety policy, transfusion-related lung injury (TRALI) continues to occur, suggesting an incomplete understanding of its pathogenesis. Objectives To evaluate the association between the composition of the red blood cell unit (RBC) and the onset of ALI after transfusion, defined by a ratio between partial pressure of oxygen (PaO 2 ) and the fraction of inspired oxygen (FiO 2 ) ≤ 300 mmHg in the first 3 days postoperatively. Methods Adults undergoing scheduled cardiac surgery at Nantes University Hospital between September 2016 and March 2021 and requiring transfusion of 1–5 RBC during surgery were included. To determine the exposure of each patient (i.e., total amount of inflammatory proteins received during transfusion), we analyzed the composition (panel of 15 proteins) of each RBC received by the participants. After stimulation of peripheral blood mononuclear cells (PBMC) with supernatant RBC (SN-RBC), NK cell cytotoxicity to pulmonary epithelial cells (Calu_3 cells) was assessed. Finally, we determined the association between SN-RBC composition, patient characteristics, and blood donation preparation methods. Results Over the study period, 161 patients were included, of whom 54 (33.5%) developed ALI within the first 3 days. Patients with ALI had a significantly higher median (IQR) exposure to SDF_1α than non-ALI patients 2.0 × 10 4 (0.0 to 4.2 × 10 4 ) vs. 1.0 × 10 4 (0.0 to 2.7 × 10 4 ) picograms, P = 0.02. In vitro, PBMC coculture with SN-RBC containing high levels of SDF_1α enhanced NKG2D-dependant NKG2A + NK cell cytotoxicity to Calu_3 cells. RBC from female donors or donors with platelet counts higher than 300 × 10 9 /L showed the highest concentration of SDF_1α, as well as RBC prepared by "whole blood filtration". Conclusions High exposure to SDF_1α through transfusion may be associated with the onset of ALI following cardiac surgery. The increase in NKG2D-dependent cytotoxicity of NKG2A + NK cells is a possible explanation for this finding. These results advocate for better characterization of the determinants of RBC composition and for developing new immune strategies to mitigate transfusion side effects.

Mucosal-associated invariant T cells (MAIT cells) mediate tissue homeostasis and antimicrobial immunity. However, the cells that express major histocompatibility complex (MHC) class I-related protein 1 (MR1) and present microbial vitamin B-derived antigens (VitBAg) to MAIT cells remain unknown. We found that MR1 expression varied across tissues and cell types. Macrophages from the lung and peritoneal cavity expressed the highest levels of MR1 and were the most efficient at capturing and presenting VitBAg to MAIT cells. Expression of MR1 in macrophages was regulated transcriptionally and induced by the tissue environment and microbiota. Depletion of MR1 in macrophages, dendritic cells, and monocytes changed the composition of the microbiota and impaired MAIT cell responses against bacterial infection. We concluded that macrophages are key for MR1 antigen presentation and MAIT cell immunity.

CD83 is critical for CD4(+) T cell selection. It regulates MHC II ubiquitination and turnover at the surface of thymic epithelial cells (TECs). The role of UBL3, a recently identified adaptor molecule for MHC II ubiquitination, is unknown in thymic selection. Here we demonstrate that UBL3 regulates MHC II in TECs and participates in CD4(+) T cell selection. Deleting UBL3 in CD83 loss-of-function mice (Cd83anu/anu Ubl3-/-) increases MHC II on the surface of Cd83anu/anu TECs. This increase in surface MHC II correlates with increased positive selection of CD4(+) T cells. Analysis of Cd83anu/anu and Cd83anu/anu Ubl3-/- mice identifies the CD4(+) CD8(low) CD69(+) stage of positive selection as the origin of the CD4(+) T cell selection defect in Cd83anu/anu mice. This stage of CD4(+) T cell positive selection is also impacted by UBL3. The positive selection defect in the absence of CD83 also manifests as alterations in CCR7(+) CD4 single-positive (SP) thymocytes. At the later stages of CD4(+) T cell development, a role for UBL3 is no longer detected. In summary, through in-depth phenotyping of thymocyte populations, a role for CD83 and UBL3 in regulating the early stages of CD4(+) T cell positive selection has been identified.

The major histocompatibility complex (MHC) class I-related protein 1 (MR1) presents vitamin B-derived metabolites to mucosal-associated invariant T (MAIT) and other T cells. There is limited polymorphism of MR1, the functional impact of which is not understood. We examined the impact of allelic variation of MR1 on the expression, structure and function of the known MR1 allomorphs. The expression and function of MR1∗02, MR1∗03, and MR1∗06 were similar to the canonical MR1∗01. Crystal structures of four MR1 allomorphs show that their polymorphisms do not impact the three-dimensional fold of MR1. Despite the binding of 5-OP-RU to MR1∗05 and its cell surface upregulation, this allomorph was severely impaired in its ability to activate primary MAIT cells. This phenotype was controlled by two (His90Gln and Glu52Gly) of its three polymorphisms, which led to the loss of structurally stabilizing interactions. When cells expressing the MR1 allomorphs were infected with herpes simplex virus type 1 (HSV-1), the nascent expression of all allomorphs was severely impaired, but surface expression of MR1∗04:01 and MR1∗04:02 was relatively less impacted. Hence, MR1 allelic variation alters the expression and function of the MR1∗04 and MR1∗05 allomorphs, with implications for MAIT cell and diverse MR1-reactive T cell immunity.

Major histocompatibility complex class I-related protein 1 (MR1) presents microbial Vitamin B-related metabolite antigens (VitBAg) at the cell surface to activate mucosal-associated invariant T (MAIT) cells. Precisely how antigen-presenting cells capture these MR1 ligands is not known. Here, we show that the most effective route for presentation of bacterial VitBAg involves passage through the cytosol. Consistent with structural similarities with riboflavin, we find that VitBAg presentation is inhibited by riboflavin. We further show that riboflavin carriers transport VitBAg into cells and enhance MR1 antigen presentation to MAIT cells. However, elimination of specific riboflavin carriers does not ablate VitBAg presentation, indicating cells possess redundant mechanisms to internalize this family of MR1 ligands. Our findings provide new insights into the intracellular pathway used by VitBAg to bind MR1 molecules and identify potential approaches to boost MR1-mediated MAIT cell responses for therapeutic benefits.

Major histocompatibility class II (MHC II) is critical for adaptive immunity. MHC II intracellular trafficking and degradation is regulated by ubiquitination. Poly-ubiquitination (Ub) of MHC II directs it away from the plasma membrane and is a critical determinant of MHC II turnover. The MHC II Ub chain has not been characterized. Here, we describe the poly-Ub chain associated with MHC II in primary murine antigen presenting cells; conventional dendritic cells (cDCs) and B cells. Analysis was conducted for endogenous murine MHC II isoforms H2-A and H2-E immunoprecipitated from primary cells. We show that ubiquitination of both I-A and I-E expressed by cDCs and B cells is dependent on the E3 Ub ligase MARCH1. Using mass spectrometry Ub chain linkage analysis and innovative Ub “clipping” proteomics we comprehensively defined the features of the MHC II poly-Ub chain. This revealed the MARCH1-dependent poly-Ub chain associated with MHC II is composed of a branched chain with K11 and K63 Ub chain linkages. This is the first description of the Ub chain linkages and architecture associated with MARCH1-mediated ubiquitination of MHC II in primary antigen presenting cells. This is important because it creates possibilities for manipulation of MHC II function in adaptive immunity.

MHC class I-related protein (MR1) presents vitamin B-based antigens (Ags) to mucosal-associated invariant T (MAIT) cells. While microbial riboflavin (RF) precursors are well-documented MR1 ligands, it is unclear whether host-generated RF catabolites influence MR1 immunity. Here, we report that RF catabolites, including 10-formylmethylflavin (FMF), lumichrome, lumiflavin, and alloxazine, bind to MR1 with moderate affinity, while RF itself binds weakly. In contrast to the MR1-upregulating microbial RF precursors, RF catabolites reduced the surface level of MR1 by inducing its retention in the endoplasmic reticulum and inhibiting exit. These RF catabolites weakly competed with vitamin B-based Ags for MR1 binding, thereby selectively inhibiting MAIT activation. The crystal structures of MR1 with RF, FMF, lumiflavin, and lumichrome show binding in the A'-pocket of MR1. Here, lumichrome formed a "flavin bond" covalent interaction with MR1-Lys43 differing from the typical Schiff base. Collectively, we identified three-ringed isoalloxazines that bind MR1 and reduce surface levels, suggesting a potential role in dampening MAIT cell immunity.

Immunity to infectious diseases is predominantly studied by measuring immune responses towards a single pathogen, although co-infections are common. In-depth mechanisms on how co-infections impact anti-viral immunity are lacking, but are highly relevant to treatment and prevention. We established a mouse model of co-infection with unrelated viruses, influenza A (IAV) and Semliki Forest virus (SFV), causing disease in different organ systems. SFV infection eight days before IAV infection results in prolonged IAV replication, elevated cytokine/chemokine levels and exacerbated lung pathology. This is associated with impaired lung IAV-specific CD8 + T cell responses, stemming from suboptimal CD8 + T cell activation and proliferation in draining lymph nodes, and dendritic cell paralysis. Prior SFV infection leads to increased blood brain barrier permeability and presence of IAV RNA in brain, associated with increased trafficking of IAV-specific CD8 + T cells and establishment of long-term tissue-resident memory. Relative to lung IAV-specific CD8 + T cells, brain memory IAV-specific CD8 + T cells have increased TCR repertoire diversity within immunodominant D b NP 366 + CD8 + and D b PA 224 + CD8 + responses, featuring suboptimal TCR clonotypes. Overall, our study demonstrates that infection with an unrelated neurotropic virus perturbs IAV-specific immune responses and exacerbates IAV disease. Our work provides key insights into therapy and vaccine regimens directed against unrelated pathogens.

Presentation of metabolites by the major histocompatibility complex class I-related - related protein 1 (MR1) molecule to mucosalassociated invariant T cells is impaired during herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infections. This is surprising given these viruses do not directly synthesise MR1 ligands. We have previously identified fi ed several HSV proteins responsible for rapidly downregulating the intracellular pool of immature MR1, effectively inhibiting new surface antigen presentation, while preexisting ligand-bound mature MR1 is unexpectedly upregulated by HSV-1. Using fl ow cytometry, immunoblotting, and high-throughput fl uorescence microscopy, we demonstrate that the endocytosis of surface MR1 is impaired during HSV infection and that internalized molecules accumulate in EEA1-labeled early endosomes, avoiding degradation. We establish that the short MR1 cytoplasmic tail is not required for HSV-1-mediated - mediated downregulation of immature molecules; however it may play a role in the retention of mature molecules on the surface and in early endosomes. We also determine that the HSV-1 US3 protein, the shorter US3.5 kinase and the full-length HSV-2 homolog, all predominantly target mature surface rather than total MR1 levels. We propose that the downregulation of intracellular and cell surface MR1 molecules by US3 and other HSV proteins is an immune-evasive countermeasure to minimize the effect of impaired MR1 endocytosis, which might otherwise render infected cells susceptible to MR1-mediated killing by mucosalassociated invariant T cells.

Summary: The major histocompatibility complex (MHC), Class-I-related (MR1) molecule presents microbiome-synthesized metabolites to Mucosal-associated invariant T (MAIT) cells, present at sites of herpes simplex virus (HSV) infection. During HSV type 1 (HSV-1) infection there is a profound and rapid loss of MR1, in part due to expression of unique short 3 protein. Here we show that virion host shutoff RNase protein downregulates MR1 protein, through loss of MR1 transcripts. Furthermore, a third viral protein, infected cell protein 22, also downregulates MR1, but not classical MHC-I molecules. This occurs early in the MR1 trafficking pathway through proteasomal degradation. Finally, HSV-2 infection results in the loss of MR1 transcripts, and intracellular and surface MR1 protein, comparable to that seen during HSV-1 infection. Thus HSV coordinates a multifaceted attack on the MR1 antigen presentation pathway, potentially protecting infected cells from MAIT cell T cell receptor-mediated detection at sites of primary infection and reactivation.

Conventional dendritic cells (cDC) are professional antigen-presenting cells able to prime naive T cells. Here, we present a protocol for ex vivo T cell priming by murine splenic cDC. We describe the steps of injecting fluorescently labeled antigens to mice, purifying antigen-bearing cDC, and priming antigen-specific T cells ex vivo. This protocol is suitable for studying the T cell priming function of cDC in various murine models and helps factor in the effect of the microenvironment on cDC ability to uptake and process antigens.For complete details on the use and execution of this protocol, please refer to Ashayeripanah et al.1

MHC antigen presentation plays a fundamental role in adaptive and semi-invariant T cell immunity. Distinct MHC molecules bind antigens that differ in chemical structure, origin and location and present them to specialized T cells. MHC class I-related protein 1 (MR1) presents a range of small molecule antigens to MR1-restricted T (MR1T) lymphocytes. The best studied MR1 ligands are derived from microbial metabolism and are recognized by a major class of MR1T cells known as mucosal-associated invariant T (MAIT) cells. Here, we describe the MR1 antigen presentation pathway: the known types of antigens presented by MR1, the location where MR1–antigen complexes form, the route followed by the complexes to the cell surface, the mechanisms involved in termination of MR1 antigen presentation and the accessory cellular proteins that comprise the MR1 antigen presentation machinery. The current road map of the MR1 antigen presentation pathway reveals potential strategies for therapeutic manipulation of MR1T cell function and provides a foundation for further studies that will lead to a deeper understanding of MR1-mediated immunity. The MHC class I-related protein 1 (MR1) presents specific small molecule antigens to MR1-restricted T (MR1T) lymphocytes. These cells play an important role in infection and cancer, and strategies to target these cells are of considerable therapeutic interest. In this Review, McWilliam and Villadangos provide a comprehensive description of the antigen presentation pathway of MR1, which is fundamental for the understanding of MR1-mediated immunity and the potential therapeutic manipulation of MR1T cells.

Dendritic cell (DC)-targeted vaccination is a new mode of antigen delivery that relies on the use of monoclonal antibodies (mAb) to target antigen to specific DC subsets. The neonatal Fc receptor (FcRn) is a non-classical Fc receptor that binds to immunoglobulin G (IgG) in acidified endosomes and controls its intracellular transport and recycling. FcRn is known to participate in the antigen presentation of immune complexes, however its contribution to DC-targeted vaccination has not previously been examined. Here we have investigated the role of FcRn in antigen presentation using antigen conjugated to IgG mAb which target specific DC receptors, including DEC205 and Clec9A expressed by the conventional DC 1 (cDC1) subset. We show that FcRn is expressed at high levels by cDC1, both at steady-state and following activation and plays a significant role in MHC I cross-presentation and MHC II presentation of antigens that are targeted to cDC1 via mAb specific for DEC205. This effect of FcRn is intrinsic to cDC1 and FcRn impacts the efficacy of anti-DEC205-mediated vaccination against B cell lymphoma. In contrast, FcRn does not impact presentation of antigens targeted to Clec9A and does not regulate presentation of cell-associated antigen. These data highlight a new and unique role of FcRn in controlling the immunogenicity of anti-DEC205-based vaccination, with consequences for exploiting this pathway to improve DC-targeted vaccine outcomes.

Sepsis induces immune alterations, which last for months after the resolution of illness. The effect of this immunological reprogramming on the risk of developing cancer is unclear. Here we use a national claims database to show that sepsis survivors had a lower cumulative incidence of cancers than matched nonsevere infection survivors. We identify a chemokine network released from sepsis-trained resident macrophages that triggers tissue residency of T cells via CCR2 and CXCR6 stimulations as the immune mechanism responsible for this decreased risk of de novo tumor development after sepsis cure. While nonseptic inflammation does not provoke this network, laminarin injection could therapeutically reproduce the protective sepsis effect. This chemokine network and CXCR6 tissue-resident T cell accumulation were detected in humans with sepsis and were associated with prolonged survival in humans with cancer. These findings identify a therapeutically relevant antitumor consequence of sepsis-induced trained immunity. Here the authors show that sepsis and its resolution alter cancer susceptibility by epigenetically altering resident macrophages resulting in retention of T cells that increase antitumoral immunity.

The development of dendritic cells (DCs) depends on signaling via the FMS-like tyrosine kinase 3 (Flt3) receptor. How Flt3 signaling impacts terminally differentiated DC function is unknown. This is important given the increasing interest in exploiting Flt3 for vaccination and tumor immunotherapy. Here, we examined DCs in mice harboring constitutively activated Flt3 (Flt3-ITD). Flt3ITD/ITD mice possessed expanded splenic DC subsets including plasmacytoid DC, conventional DC (cDC)1, cDC2, double positive (DP) cDC1 (CD11c+ CD8+ CD11b- CD103+ CD86+), noncanonical (NC) cDC1 (CD11c+ CD8+ CD11b- CD103- CD86-) and single positive (SP) cDC1 (CD11c+ CD8+ CD11b- CD103- CD86+). Outcomes of constitutive Flt3 signaling differed depending on the cDC subset examined. In comparison with wild type (WT) DCs, all Flt3ITD/ITD cDCs displayed an altered surface phenotype with changes in costimulatory molecules, major histocompatibility complex class I (MHC I) and II (MHC II). Cytokine secretion patterns, antigen uptake, antigen proteolysis and antigen presenting function differed between WT and Flt3ITD/ITD subsets, particularly cDC2. In summary, Flt3 signaling impacts the function of terminally differentiated cDCs with important consequences for antigen presentation.

Bacterial synthesis of vitamin B2 generates a by-product, 5-(2-oxopropylideneamino)- d -ribityl-aminouracil (5-OP-RU), with potent immunological properties in mammals, but it is rapidly degraded in water. This natural product covalently bonds to the key immunological protein MR1 in the endoplasmic reticulum of antigen presenting cells (APCs), enabling MR1 refolding and trafficking to the cell surface, where it interacts with T cell receptors (TCRs) on mucosal associated invariant T lymphocytes (MAIT cells), activating their immunological and antimicrobial properties. Here, we strategically modify this natural product to understand the molecular basis of its recognition by MR1. This culminated in the discovery of new water-stable compounds with extremely powerful and distinctive immunological functions. We report their capacity to bind MR1 inside APCs, triggering its expression on the cell surface (EC 50 17 nM), and their potent activation (EC 50 56 pM) or inhibition (IC 50 80 nM) of interacting MAIT cells. We further derivatize compounds with diazirine-alkyne, biotin, or fluorophore (Cy5 or AF647) labels for detecting, monitoring, and studying cellular MR1. Computer modeling casts new light on the molecular mechanism of activation, revealing that potent activators are first captured in a tyrosine- and serine-lined cleft in MR1 via specific pi-interactions and H-bonds, before more tightly attaching via a covalent bond to Lys43 in MR1. This chemical study advances our molecular understanding of how bacterial metabolites are captured by MR1, influence cell surface expression of MR1, interact with T cells to induce immunity, and offers novel clues for developing new vaccine adjuvants, immunotherapeutics, and anticancer drugs.