TMPRSS2 is a functional receptor for human coronavirus HKU1.

Four endemic seasonal human coronaviruses causing common colds circulate worldwide: HKU1, 229E, NL63 and OC43 (ref. 1). After binding to cellular receptors, coronavirus spike proteins are primed for fusion by transmembrane serine protease 2 (TMPRSS2) or endosomal cathepsins2-9. NL63 uses angiotensin-converting enzyme 2 as a receptor10, whereas 229E uses human aminopeptidase-N11. HKU1 and OC43 spikes bind cells through 9-O-acetylated sialic acid, but their protein receptors remain unknown12. Here we show that TMPRSS2 is a functional receptor for HKU1. TMPRSS2 triggers HKU1 spike-mediated cell-cell fusion and pseudovirus infection. Catalytically inactive TMPRSS2 mutants do not cleave HKU1 spike but allow pseudovirus infection. Furthermore, TMPRSS2 binds with high affinity to the HKU1 receptor binding domain (Kd 334 and 137 nM for HKU1A and HKU1B genotypes) but not to SARS-CoV-2. Conserved amino acids in the HKU1 receptor binding domain are essential for binding to TMPRSS2 and pseudovirus infection. Newly designed anti-TMPRSS2 nanobodies potently inhibit HKU1 spike attachment to TMPRSS2, fusion and pseudovirus infection. The nanobodies also reduce infection of primary human bronchial cells by an authentic HKU1 virus. Our findings illustrate the various evolution strategies of coronaviruses, which use TMPRSS2 to either directly bind to target cells or prime their spike for membrane fusion and entry.

The CD46-Jagged1 interaction is critical for human TH1 immunity.

CD46 is a complement regulator with important roles related to the immune response. CD46 functions as a pathogen receptor and is a potent costimulator for the induction of interferon-γ (IFN-γ)-secreting effector T helper type 1 (T(H)1) cells and their subsequent switch into interleukin 10 (IL-10)-producing regulatory T cells. Here we identified the Notch family member Jagged1 as a physiological ligand for CD46. Furthermore, we found that CD46 regulated the expression of Notch receptors and ligands during T cell activation and that disturbance of the CD46-Notch crosstalk impeded induction of IFN-γ and switching to IL-10. Notably, CD4(+) T cells from CD46-deficient patients and patients with hypomorphic mutations in the gene encoding Jagged1 (Alagille syndrome) failed to mount appropriate T(H)1 responses in vitro and in vivo, which suggested that CD46-Jagged1 crosstalk is responsible for the recurrent infections in subpopulations of these patients.

The Inhibitory Receptor NKG2A Sustains Virus-Specific CD8⁺ T Cells in Response to a Lethal Poxvirus Infection.

CD8(+) T cells and NK cells protect from viral infections by killing virally infected cells and secreting interferon-γ. Several inhibitory receptors limit the magnitude and duration of these anti-viral responses. NKG2A, which is encoded by Klrc1, is a lectin-like inhibitory receptor that is expressed as a heterodimer with CD94 on NK cells and activated CD8(+) T cells. Previous studies on the impact of CD94/NKG2A heterodimers on anti-viral responses have yielded contrasting results and the in vivo function of NKG2A remains unclear. Here, we generated Klrc1(-/-) mice and found that NKG2A is selectively required for resistance to ectromelia virus (ECTV). NKG2A functions intrinsically within ECTV-specific CD8(+) T cells to limit excessive activation, prevent apoptosis, and preserve the specific CD8(+) T cell response. Thus, although inhibitory receptors often cause T cell exhaustion and viral spreading during chronic viral infections, NKG2A optimizes CD8(+) T cell responses during an acute poxvirus infection.

Complement Regulates Nutrient Influx and Metabolic Reprogramming during Th1 Cell Responses.

Expansion and acquisition of Th1 cell effector function requires metabolic reprogramming; however, the signals instructing these adaptations remain poorly defined. Here we found that in activated human T cells, autocrine stimulation of the complement receptor CD46, and specifically its intracellular domain CYT-1, was required for induction of the amino acid (AA) transporter LAT1 and enhanced expression of the glucose transporter GLUT1. Furthermore, CD46 activation simultaneously drove expression of LAMTOR5, which mediated assembly of the AA-sensing Ragulator-Rag-mTORC1 complex and increased glycolysis and oxidative phosphorylation (OXPHOS), required for cytokine production. T cells from CD46-deficient patients, characterized by defective Th1 cell induction, failed to upregulate the molecular components of this metabolic program as well as glycolysis and OXPHOS, but IFN-γ production could be reinstated by retrovirus-mediated CD46-CYT-1 expression. These data establish a critical link between the complement system and immunometabolic adaptations driving human CD4(+) T cell effector function.

Development of a highly specific and sensitive VHH-based sandwich immunoassay for the detection of the SARS-CoV-2 nucleoprotein.

The current COVID-19 pandemic illustrates the importance of obtaining reliable methods for the rapid detection of SARS-CoV-2. A highly specific and sensitive diagnostic test able to differentiate the SARS-CoV-2 virus from common human coronaviruses is therefore needed. Coronavirus nucleoprotein (N) localizes to the cytoplasm and the nucleolus and is required for viral RNA synthesis. N is the most abundant coronavirus protein, so it is of utmost importance to develop specific antibodies for its detection. In this study, we developed a sandwich immunoassay to recognize the SARS-CoV-2 N protein. We immunized one alpaca with recombinant SARS-CoV-2 N and constructed a large single variable domain on heavy chain (VHH) antibody library. After phage display selection, seven VHHs recognizing the full N protein were identified by ELISA. These VHHs did not recognize the nucleoproteins of the four common human coronaviruses. Hydrogen Deuterium eXchange-Mass Spectrometry (HDX-MS) analysis also showed that these VHHs mainly targeted conformational epitopes in either the C-terminal or the N-terminal domains. All VHHs were able to recognize SARS-CoV-2 in infected cells or on infected hamster tissues. Moreover, the VHHs could detect the SARS variants B.1.17/alpha, B.1.351/beta, and P1/gamma. We propose that this sandwich immunoassay could be applied to specifically detect the SARS-CoV-2 N in human nasal swabs.

Complement regulator CD46 temporally regulates cytokine production by conventional and unconventional T cells.

In this study we demonstrate a new form of immunoregulation: engagement on CD4(+) T cells of the complement regulator CD46 promoted the effector potential of T helper type 1 cells (T(H)1 cells), but as interleukin 2 (IL-2) accumulated, it switched cells toward a regulatory phenotype, attenuating IL-2 production via the transcriptional regulator ICER/CREM and upregulating IL-10 after interaction of the CD46 tail with the serine-threonine kinase SPAK. Activated CD4(+) T cells produced CD46 ligands, and blocking CD46 inhibited IL-10 production. Furthermore, CD4(+) T cells in rheumatoid arthritis failed to switch, consequently producing excessive interferon-gamma (IFN-gamma). Finally, gammadelta T cells, which rarely produce IL-10, expressed an alternative CD46 isoform and were unable to switch. Nonetheless, coengagement of T cell antigen receptor (TCR) gammadelta and CD46 suppressed effector cytokine production, establishing that CD46 uses distinct mechanisms to regulate different T cell subsets during an immune response.

Intracellular complement activation sustains T cell homeostasis and mediates effector differentiation.

Complement is viewed as a critical serum-operative component of innate immunity, with processing of its key component, C3, into activation fragments C3a and C3b confined to the extracellular space. We report here that C3 activation also occurred intracellularly. We found that the T cell-expressed protease cathepsin L (CTSL) processed C3 into biologically active C3a and C3b. Resting T cells contained stores of endosomal and lysosomal C3 and CTSL and substantial amounts of CTSL-generated C3a. While "tonic" intracellular C3a generation was required for homeostatic T cell survival, shuttling of this intracellular C3-activation-system to the cell surface upon T cell stimulation induced autocrine proinflammatory cytokine production. Furthermore, T cells from patients with autoimmune arthritis demonstrated hyperactive intracellular complement activation and interferon-γ production and CTSL inhibition corrected this deregulated phenotype. Importantly, intracellular C3a was observed in all examined cell populations, suggesting that intracellular complement activation might be of broad physiological significance.

The "ins and outs" of complement-driven immune responses.

The complement system represents an evolutionary old and critical component of innate immunity where it forms the first line of defense against invading pathogens. Originally described as a heat-labile fraction of the serum responsible for the opsonization and subsequent lytic killing of bacteria, work over the last century firmly established complement as a key mediator of the general inflammatory response but also as an acknowledged vital bridge between innate and adaptive immunity. However, recent studies particularly spanning the last decade have provided new insights into the novel modes and locations of complement activation and highlighted unexpected additional biological functions for this ancient system, for example, in regulating basic processes of the cell. In this review, we will cover the current knowledge about complement's established and novel roles in innate and adaptive immunity with a focus on the functional differences between serum circulating and intracellularly active complement and will describe and discuss the newly discovered cross-talks of complement with other cell effector systems particularly during T-cell induction and contraction.

Dysregulated CD46 shedding interferes with Th1-contraction in systemic lupus erythematosus.

IFN-γ-producing T helper 1 (Th1) cell responses mediate protection against infections but uncontrolled Th1 activity also contributes to a broad range of autoimmune diseases. Autocrine complement activation has recently emerged as key in the induction and contraction of human Th1 immunity: activation of the complement regulator CD46 and the C3aR expressed by CD4+ T cells via autocrine generated ligands C3b and C3a, respectively, are critical to IFN-γ production. Further, CD46-mediated signals also induce co-expression of immunosuppressive IL-10 in Th1 cells and transition into a (self)-regulating and contracting phase. In consequence, C3 or CD46-deficient patients suffer from recurrent infections while dysregulation of CD46 signaling contributes to Th1 hyperactivity in rheumatoid arthritis and multiple sclerosis. Here, we report a defect in CD46-regulated Th1 contraction in patients with systemic lupus erythematosus (SLE). We observed that MMP-9-mediated increased shedding of soluble CD46 by Th1 cells was associated with this defect and that inhibition of MMP-9 activity normalized release of soluble CD46 and restored Th1 contraction in patients' T cells. These data may deliver the first mechanistic explanation for the increased serum CD46 levels observed in SLE patients and indicate that targeting CD46-cleaving proteases could be a novel avenue to modulate Th1 responses.

The role of complement in CD4⁺ T cell homeostasis and effector functions.

The complement system is among the evolutionary oldest 'players' of the immune system. It was discovered in 1896 by Jules Bordet as a heat-labile fraction of the serum responsible for the opsonisation and subsequent killing of bacteria. The decades between the 1920s and 1990s then marked the discovery and biochemical characterization of the proteins comprising the complement system. Today, complement is defined as a complex system consisting of more than 30 membrane-bound and soluble plasma proteins, which are activated in a cascade-like manner, very similarly to the caspase proteases and blood coagulation systems. Complement is engrained in the immunologist's mind as a serum-effective, quintessential part of innate immunity, vitally required for the detection and removal of pathogens or other dangerous entities. Three decades ago, this rather confined definition was challenged and then refined when it was shown that complement participates vitally in the induction and regulation of B cell responses, thus adaptive immunity. Similarly, research work published in more recent years supports an equally important role for the complement system in shaping T cell responses. Today, we are again facing paradigm shifts in the field: complement is actively involved in the negative control of T cell effector immune responses, and thus, by definition in immune homeostasis. Further, while serum complement activity is without doubt fundamental in the defence against invading pathogens, local immune cell-derived production of complement emerges as key mediator of complement's impact on adaptive immune responses. And finally, the impact of complement on metabolic pathways and the crosstalk between complement and other immune effector systems is likely more extensive than previously anticipated and is fertile ground for future discoveries. In this review, we will discuss these emerging new roles of complement, with a focus on Th1 cell biology.

C3a modulates IL-1ß secretion in human monocytes by regulating ATP efflux and subsequent NLRP3 inflammasome activation.

Interleukin-1ß (IL-1ß) is a proinflammatory cytokine and a therapeutic target in several chronic autoimmune states. Monocytes and macrophages are the major sources of IL-1ß. IL-1ß production by these cells requires Toll-like receptor (TLR) and adenosine triphosphate (ATP)-mediated P2X purinoceptor 7 (P2X7) signals, which together activate the inflammasome. However, how TLR signals and ATP availability are regulated during monocyte activation is unclear and the involvement of another danger signal system has been proposed. Here, we demonstrate that both lipopolysaccharide (LPS) and the anaphylatoxin C3a are needed for IL-1ß production in human macrophages and dendritic cells, while in monocytes, C3a enhanced the secretion of LPS-induced IL-1ß. C3a and LPS-stimulated monocytes increased T helper 17 (Th17) cell induction in vitro, and human rejecting, but not nonrejecting, kidney transplant biopsies were characterized by local generation of C3a and monocyte and Th17 cell infiltration. Mechanistically, C3a drives IL-1ß production in monocytes by controlling the release of intracellular ATP into the extracellular space via regulation of as-yet unidentified ATP-releasing channels in an extracellular signal-regulated kinase 1/2-dependent fashion. These data define a novel function for complement in inflammasome activation in monocytes and suggest that C3aR-mediated signaling is a vital component of the IL-1ß-Th17 axis.

The Th1 life cycle: molecular control of IFN-γ to IL-10 switching.

Control of IFN-γ-secreting T helper (Th) 1 cells prevents autoimmunity and immunopathology during infection. IL-10-mediated suppression of Th1 cells is achieved not only through IL-10 produced extrinsically, but also through a negative feedback loop that induces "intrinsic" IL-10 expression in cells also expressing IFN-γ, during Th1 lineage differentiation. Targeting this Th1 cell IFN-γ to IL-10 switching is a tantalising prospect for developing therapeutics for Th1-mediated diseases. In this review, the molecular pathways that regulate IFN-γ versus IL-10 expression in Th1 cells are examined, with focus on the role of complement regulator and T cell co-stimulatory molecule CD46, and also discussed are challenges and controversies in the field.

Mouse PRDM9 DNA-binding specificity determines sites of histone H3 lysine 4 trimethylation for initiation of meiotic recombination.

Meiotic recombination generates reciprocal exchanges between homologous chromosomes (also called crossovers, COs) that are essential for proper chromosome segregation during meiosis and are a major source of genome diversity by generating new allele combinations. COs have two striking properties: they occur at specific sites, called hotspots, and these sites evolve rapidly. In mammals, the Prdm9 gene, which encodes a meiosis-specific histone H3 methyltransferase, has recently been identified as a determinant of CO hotspots. Here, using transgenic mice, we show that the sole modification of PRDM9 zinc fingers leads to changes in hotspot activity, histone H3 lysine 4 trimethylation (H3K4me3) levels, and chromosome-wide distribution of COs. We further demonstrate by an in vitro assay that the PRDM9 variant associated with hotspot activity binds specifically to DNA sequences located at the center of the three hotspots tested. Remarkably, we show that mutations in cis located at hotspot centers and associated with a decrease of hotspot activity affect PRDM9 binding. Taken together, these results provide the direct demonstration that Prdm9 is a master regulator of hotspot localization through the DNA binding specificity of its zinc finger array and that binding of PRDM9 at hotspots promotes local H3K4me3 enrichment.

T cell migration and effector function differences in familial adenomatous polyposis patients with APC gene mutations.

Familial adenomatous polyposis (FAP) is an inherited disease characterized by the development of large number of colorectal adenomas with high risk of evolving into colorectal tumors. Mutations of the Adenomatous polyposis coli (APC) gene is often at the origin of this disease, as well as of a high percentage of spontaneous colorectal tumors. APC is therefore considered a tumor suppressor gene. While the role of APC in intestinal epithelium homeostasis is well characterized, its importance in immune responses remains ill defined. Our recent work indicates that the APC protein is involved in various phases of both CD4 and CD8 T cells responses. This prompted us to investigate an array of immune cell features in FAP subjects carrying APC mutations. A group of 12 FAP subjects and age and sex-matched healthy controls were studied. We characterized the immune cell repertoire in peripheral blood and the capacity of immune cells to respond ex vivo to different stimuli either in whole blood or in purified T cells. A variety of experimental approaches were used, including, pultiparamater flow cytometry, NanosString gene expression profiling, Multiplex and regular ELISA, confocal microscopy and computer-based image analyis methods. We found that the percentage of several T and natural killer (NK) cell populations, the expression of several genes induced upon innate or adaptive immune stimulation and the production of several cytokines and chemokines was different. Moreover, the capacity of T cells to migrate in response to chemokine was consistently altered. Finally, immunological synapses between FAP cytotoxic T cells and tumor target cells were more poorly structured. Our findings of this pilot study suggest that mild but multiple immune cell dysfunctions, together with intestinal epithelial dysplasia in FAP subjects, may facilitate the long-term polyposis and colorectal tumor development. Although at an initial discovery phase due to the limited sample size of this rare disease cohort, our findings open new perspectives to consider immune cell abnormalities into polyposis pathology.

DAP10 associates with Ly49 receptors but contributes minimally to their expression and function in vivo.

NK cells recognize target cells through activating receptors, many of which rely on the transmembrane adaptors DAP10, DAP12 and FcR-gamma to deliver intracellular signals. Because these adaptors initiate distinct signaling pathways, they dictate the type of response mediated by receptor engagement. DAP10, for example, primarily triggers cytotoxicity, whereas DAP12 induces both cytotoxicity and IFN-gamma secretion. In mice, NKG2D signals through both DAP10 and DAP12, which broadens and modulates the type of response engendered by encounter with ligand. Although initial studies indicated that Ly49H and Ly49D recruit only DAP12, a recent report suggested that they also associate with DAP10. We asked whether this association occurs and is functionally significant under physiologic conditions. Our data demonstrate that DAP10 does associate with Ly49H and Ly49D in primary NK cells. While this association contributes slightly to cell surface expression of both receptors, it has no significant impact on Ly49H-mediated control of murine cytomegalovirus infection. Thus, while many activating NK-cell receptors are promiscuous in terms of adaptor association, our data indicate that the functional consequences of such promiscuity may vary widely and may not be evident in all cases.

DNAX accessory molecule-1 (CD226) promotes human hepatocellular carcinoma cell lysis by Vgamma9Vdelta2 T cells.

Human Vgamma9Vdelta2 T lymphocytes can be activated by nonpeptidic antigens such as the mevalonate pathway-derived isopentenyl pyrophosphate or synthetic phosphoantigen such as bromohydrin pyrophosphate. They display a strong cytotoxic activity against several tumor types, including hepatocellular carcinoma (HCC). Little is known about the mechanisms underlying Vgamma9Vdelta2 T-cell recognition of tumor cells, but there is strong evidence that activating NK receptors play a role in gammadelta T-cell cytotoxicity. In this study, we showed that the two NK receptors DNAX accessory molecule-1 (DNAM-1) and CD96 were expressed by Vgamma9Vdelta2 T cells. The ligands Nectin-like-5 specific of both DNAM-1 and CD96, and also Nectin-2, an additional ligand of DNAM-1, were present on all HCC cell lines analyzed. Furthermore, we demonstrated by mAb-mediated masking experiments that cytotoxicity against HCC cells as well as IFN-gamma production in gammadelta T cells were dependent on DNAM-1. Our experiments indicated that Nectin-like-5 but not Nectin-2 was involved in DNAM-1-dependent gammadelta T-cell functions. We did not reveal a role for CD96 in the killing of HCC cells. Finally, we showed by combined mAb-mediated blockade that DNAM-1 and NKG2D could cooperate in the cell lysis of HCC.

Clinical relevance of soluble HLA class I molecules in Waldenstrom Macroglobulinemia.

OBJECTIVES: Waldenstrom Macroglobulinemia (WM) is a B-cell neoplasm characterised by secretion of IgM by lymphoplasmacytic bone marrow cells and by cytopenias and hypogammaglobulinemia in a subset of patients. Beta-2 microglobulin (b2m) is a major prognostic factor in WM and the heavy chain of HLA class I molecules, which are known to have immunosuppressive properties and have been implicated in the pathogeny of several malignancies. METHODS: We assessed the serum levels of the total soluble HLA-I molecules and the HLA-Gs molecules in 105 patients with IgM-related disorders [WM (n = 42) and IgM MGUS (n = 63)], and compared the results to 41 healthy subjects. RESULTS: We found higher levels of HLA-Is in WM, compared to IgM MGUS and healthy donors. HLA-Gs levels were similar in WM and in IgM MGUS, but higher than in healthy donors. The association between HLA-Is at the cut-off of 1.8 microg/mL and known markers of poor prognosis was then evaluated among WM patients using univariate and multivariate methods. Based on this, high HLA-Is level was strongly associated with high serum beta2M level >3 mg/L [OR = 2, (CI 95% 1.1-5.7); P = 0.04], age > 65 yrs [OR = 1.5, (CI 95% 0.5-4.1), P = 0.06] and haemoglobin < or =11.5 g/dL [OR = 3.3, (CI 95% 1.2-9.7); P = 0.03]. High levels of serum HLA-Is were also found in patients with cryoglobulinemia, however irrespectively of WM or IgM-MGUS status. CONCLUSION: Together our results suggest a possible role for soluble MHC class I molecules in WM disease. Further investigations are necessary to further demonstrate the prognostic impact of soluble MHC class I molecules in Waldenstrom Macroglobulinemia.

Complement receptor CD46 co-stimulates optimal human CD8+ T cell effector function via fatty acid metabolism.

The induction of human CD4+ Th1 cells requires autocrine stimulation of the complement receptor CD46 in direct crosstalk with a CD4+ T cell-intrinsic NLRP3 inflammasome. However, it is unclear whether human cytotoxic CD8+ T cell (CTL) responses also rely on an intrinsic complement-inflammasome axis. Here we show, using CTLs from patients with CD46 deficiency or with constitutively-active NLRP3, that CD46 delivers co-stimulatory signals for optimal CTL activity by augmenting nutrient-influx and fatty acid synthesis. Surprisingly, although CTLs express NLRP3, a canonical NLRP3 inflammasome is not required for normal human CTL activity, as CTLs from patients with hyperactive NLRP3 activity function normally. These findings establish autocrine complement and CD46 activity as integral components of normal human CTL biology, and, since CD46 is only present in humans, emphasize the divergent roles of innate immune sensors between mice and men.

Total soluble HLA class I and soluble HLA-G in multiple myeloma and monoclonal gammopathy of undetermined significance.

Serum beta2-microglobulin, the light chain of the HLA class I molecular complex, remains one of the best survival prognostic factors in multiple myeloma, but other HLA class I molecules might be of interest in monoclonal gammopathies. In this study, we evaluate total soluble HLA class I (HLA-Is) and soluble HLA-G (HLA-Gs) in 103 patients with newly diagnosed multiple myeloma, 30 patients with monoclonal gammopathy of undetermined significance (MGUS), and 30 healthy subjects, studying their prognostic value in multiple myeloma. In multiple myeloma patients, HLA-Is and HLA-Gs median values were 0.8 microg/mL and 28 ng/mL, respectively. Median HLA-Is concentration was higher in stage II and III multiple myeloma patients than in stage I multiple myeloma, MGUS, and control patients. Median HLA-Gs was significantly lower in healthy controls than in MGUS and multiple myeloma patients. A high level of HLA-Is (> or =2.1 microg/mL) was predictive of short survival (P = 0.017). For each given level of beta2-microglobulin, the relative risk of death was higher for patients with HLA-Is > or = 2.1 microg/mL than in patients with a lower level (P = 0.047). HLA-Gs, a marker of monoclonal gammopathy, was of no prognostic value, but the addition of HLA-Is to beta2-microglobulin produced an efficient prognostic score (P < 0.0001). HLA-Is is a new marker of multiple myeloma tumor load and provides additional survival prognostic information to beta2-microglobulin.