The prostaglandin D2 antagonist asapiprant ameliorates clinical severity in young hosts infected with invasive Streptococcus pneumoniae.

Streptococcus pneumoniae (pneumococcus) remains a serious cause of pulmonary and systemic infections globally, and host-directed therapies are lacking. The aim of this study was to test the therapeutic efficacy of asapiprant, an inhibitor of prostaglandin D2 signaling, against pneumococcal infection. Treatment of young mice with asapiprant after pulmonary infection with invasive pneumococci significantly reduced systemic spread, disease severity, and host death. Protection was specific against bacterial dissemination from the lung to the blood but had no effect on pulmonary bacterial burden. Asapiprant-treated mice had enhanced antimicrobial activity in circulating neutrophils, elevated levels of reactive oxygen species (ROS) in lung macrophages/monocytes, and improved pulmonary barrier integrity indicated by significantly reduced diffusion of fluorescein isothiocyanate (FITC)-dextran from lungs into the circulation. These findings suggest that asapiprant protects the host against pneumococcal dissemination by enhancing the antimicrobial activity of immune cells and maintaining epithelial/endothelial barrier integrity in the lungs.

Identification of the KIF18A alpha-4 helix as a therapeutic target for chromosomally unstable tumor cells.

Background: The mitotic kinesin, KIF18A, is required for proliferation of cancer cells that exhibit chromosome instability (CIN), implicating it as a promising target for treatment of a subset of aggressive tumor types. Determining regions of the KIF18A protein to target for inhibition will be important for the design and optimization of effective small molecule inhibitors. Methods: In this study, we used cultured cell models to investigate the effects of mutating S284 within the alpha-4 helix of KIF18A, which was previously identified as a phosphorylated residue. Results: Mutations in S284 cause relocalization of KIF18A from the plus-ends of spindle microtubules to the spindle poles. Furthermore, KIF18A S284 mutants display loss of KIF18A function and fail to support proliferation in CIN tumor cells. Interestingly, similar effects on KIF18A localization and function were seen after treatment of CIN cells with KIF18A inhibitory compounds that are predicted to interact with residues within the alpha-4 helix. Conclusion: These data implicate the KIF18A alpha-4 helix as an effective target for inhibition and demonstrate that small molecules targeting KIF18A selectively limit CIN tumor cell proliferation and result in phenotypically similar effects on mitosis at the single cell level compared to genetic perturbations.

Identification of the KIF18A alpha-4 helix as a therapeutic target for chromosomally unstable tumor cells.

The mitotic kinesin, KIF18A, is required for proliferation of cancer cells that exhibit chromosome instability (CIN), implicating it as a promising target for treatment of a subset of aggressive tumor types. Determining regions of the KIF18A protein to target for inhibition will be important for the design and optimization of effective small molecule inhibitors. In this study, we investigated the effects of mutating S284 within the alpha-4 helix of KIF18A, which was previously identified as a phosphorylated residue. Mutations in S284 cause relocalization of KIF18A from the plus-ends of spindle microtubules to the spindle poles. Furthermore, KIF18A S284 mutants display loss of KIF18A function and fail to support proliferation in CIN tumor cells. Interestingly, similar effects on KIF18A localization and function were seen after treatment of CIN cells with KIF18A inhibitory compounds that are predicted to interact with residues within the alpha-4 helix. These data implicate the KIF18A alpha-4 helix as an effective target for inhibition and demonstrate that small molecules targeting KIF18A selectively limit CIN tumor cell proliferation and result in phenotypically similar effects on mitosis at the single cell level compared to genetic perturbations.

Eicosanoid signalling blockade protects middle-aged mice from severe COVID-19.

Coronavirus disease 2019 (COVID-19) is especially severe in aged populations1. Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are highly effective, but vaccine efficacy is partly compromised by the emergence of SARS-CoV-2 variants with enhanced transmissibility2. The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially for aged populations. Here we describe the isolation of highly virulent mouse-adapted viruses and use them to test a new therapeutic drug in infected aged animals. Many of the alterations observed in SARS-CoV-2 during mouse adaptation (positions 417, 484, 493, 498 and 501 of the spike protein) also arise in humans in variants of concern2. Their appearance during mouse adaptation indicates that immune pressure is not required for selection. For murine SARS, for which severity is also age dependent, elevated levels of an eicosanoid (prostaglandin D2 (PGD2)) and a phospholipase (phospholipase A2 group 2D (PLA2G2D)) contributed to poor outcomes in aged mice3,4. mRNA expression of PLA2G2D and prostaglandin D2 receptor (PTGDR), and production of PGD2 also increase with ageing and after SARS-CoV-2 infection in dendritic cells derived from human peripheral blood mononuclear cells. Using our mouse-adapted SARS-CoV-2, we show that middle-aged mice lacking expression of PTGDR or PLA2G2D are protected from severe disease. Furthermore, treatment with a PTGDR antagonist, asapiprant, protected aged mice from lethal infection. PTGDR antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, suggesting that the PLA2G2D-PGD2/PTGDR pathway is a useful target for therapeutic interventions.

Eicosanoid signaling as a therapeutic target in middle-aged mice with severe COVID-19.

Coronavirus disease 2019 (COVID-19) is especially severe in aged populations1. Resolution of the COVID-19 pandemic has been advanced by the recent development of SARS-CoV-2 vaccines, but vaccine efficacy is partly compromised by the recent emergence of SARS-CoV-2 variants with enhanced transmissibility2. The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially in aged populations. Here, we describe the isolation of a new set of highly virulent mouse-adapted viruses and use them to test a novel therapeutic drug useful in infections of aged animals. Initially, we show that many of the mutations observed in SARS-CoV-2 during mouse adaptation (at positions 417, 484, 501 of the spike protein) also arise in humans in variants of concern (VOC)2. Their appearance during mouse adaptation indicates that immune pressure is not required for their selection. Similar to the human infection, aged mice infected with mouse-adapted SARS-CoV-2 develop more severe disease than young mice. In murine SARS, in which severity is also age-dependent, we showed that elevated levels of an eicosanoid, prostaglandin D2 (PGD2) and of a phospholipase, PLA2G2D, contributed to poor outcomes in aged mice3,4. Using our virulent mouse-adapted SARS-CoV-2, we show that infection of middle-aged mice lacking expression of DP1, a PGD2 receptor, or PLA2G2D are protected from severe disease. Further, treatment with a DP1 antagonist, asapiprant, protected aged mice from a lethal infection. DP1 antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, and demonstrates that the PLA2G2D-PGD2/DP1 pathway is a useful target for therapeutic interventions.

TGF-ß1 potentiates Vγ9Vδ2 T cell adoptive immunotherapy of cancer.

Despite its role in cancer surveillance, adoptive immunotherapy using γδ T cells has achieved limited efficacy. To enhance trafficking to bone marrow, circulating Vγ9Vδ2 T cells are expanded in serum-free medium containing TGF-ß1 and IL-2 (γδ[T2] cells) or medium containing IL-2 alone (γδ[2] cells, as the control). Unexpectedly, the yield and viability of γδ[T2] cells are also increased by TGF-ß1, when compared to γδ[2] controls. γδ[T2] cells are less differentiated and yet display increased cytolytic activity, cytokine release, and antitumor activity in several leukemic and solid tumor models. Efficacy is further enhanced by cancer cell sensitization using aminobisphosphonates or Ara-C. A number of contributory effects of TGF-ß are described, including prostaglandin E2 receptor downmodulation, TGF-ß insensitivity, and upregulated integrin activity. Biological relevance is supported by the identification of a favorable γδ[T2] signature in acute myeloid leukemia (AML). Given their enhanced therapeutic activity and compatibility with allogeneic use, γδ[T2] cells warrant evaluation in cancer immunotherapy.

Evaluation of in vitro Assays to Assess the Modulation of Dendritic Cells Functions by Therapeutic Antibodies and Aggregates.

Therapeutic antibodies have the potential to induce immunogenicity leading to the development of anti-drug antibodies (ADA) that consequently may result in reduced serum drug concentrations, a loss of efficacy or potential hypersensitivity reactions. Among other factors, aggregated antibodies have been suggested to promote immunogenicity, thus enhancing ADA production. Dendritic cells (DC) are the most efficient antigen-presenting cell population and are crucial for the initiation of T cell responses and the subsequent generation of an adaptive immune response. This work focuses on the development of predictive in vitro assays that can monitor DC maturation, in order to determine whether drug products have direct DC stimulatory capabilities. To this end, four independent laboratories aligned a common protocol to differentiate human monocyte-derived DC (moDC) that were treated with either native or aggregated preparations of infliximab, natalizumab, adalimumab, or rituximab. These drug products were subjected to different forms of physical stress, heat and shear, resulting in aggregation and the formation of subvisible particles. Each partner developed and optimized assays to monitor diverse end-points of moDC maturation: measuring the upregulation of DC activation markers via flow cytometry, analyzing cytokine, and chemokine production via mRNA and protein quantification and identifying cell signaling pathways via quantification of protein phosphorylation. These study results indicated that infliximab, with the highest propensity to form aggregates when heat-stressed, induced a marked activation of moDC as measured by an increase in CD83 and CD86 surface expression, IL-1ß, IL-6, IL-8, IL-12, TNFα, CCL3, and CCL4 transcript upregulation and release of respective proteins, and phosphorylation of the intracellular signaling proteins Syk, ERK1/2, and Akt. In contrast, natalizumab, which does not aggregate under these stress conditions, induced no DC activation in any assay system, whereas adalimumab or rituximab aggregates induced only slight parameter variation. Importantly, the data generated in the different assay systems by each partner site correlated and supported the use of these assays to monitor drug-intrinsic propensities to drive maturation of DC. This moDC assay is also a valuable tool as an in vitro model to assess the intracellular mechanisms that drive DC activation by aggregated therapeutic proteins.

Predicting CD4 T-cell epitopes based on antigen cleavage, MHCII presentation, and TCR recognition.

Accurate predictions of T-cell epitopes would be useful for designing vaccines, immunotherapies for cancer and autoimmune diseases, and improved protein therapies. The humoral immune response involves uptake of antigens by antigen presenting cells (APCs), APC processing and presentation of peptides on MHC class II (pMHCII), and T-cell receptor (TCR) recognition of pMHCII complexes. Most in silico methods predict only peptide-MHCII binding, resulting in significant over-prediction of CD4 T-cell epitopes. We present a method, ITCell, for prediction of T-cell epitopes within an input protein antigen sequence for given MHCII and TCR sequences. The method integrates information about three stages of the immune response pathway: antigen cleavage, MHCII presentation, and TCR recognition. First, antigen cleavage sites are predicted based on the cleavage profiles of cathepsins S, B, and H. Second, for each 12-mer peptide in the antigen sequence we predict whether it will bind to a given MHCII, based on the scores of modeled peptide-MHCII complexes. Third, we predict whether or not any of the top scoring peptide-MHCII complexes can bind to a given TCR, based on the scores of modeled ternary peptide-MHCII-TCR complexes and the distribution of predicted cleavage sites. Our benchmarks consist of epitope predictions generated by this algorithm, checked against 20 peptide-MHCII-TCR crystal structures, as well as epitope predictions for four peptide-MHCII-TCR complexes with known epitopes and TCR sequences but without crystal structures. ITCell successfully identified the correct epitopes as one of the 20 top scoring peptides for 22 of 24 benchmark cases. To validate the method using a clinically relevant application, we utilized five factor VIII-specific TCR sequences from hemophilia A subjects who developed an immune response to factor VIII replacement therapy. The known HLA-DR1-restricted factor VIII epitope was among the six top-scoring factor VIII peptides predicted by ITCall to bind HLA-DR1 and all five TCRs. Our integrative approach is more accurate than current single-stage epitope prediction algorithms applied to the same benchmarks. It is freely available as a web server (http://salilab.org/itcell).

T cells from hemophilia A subjects recognize the same HLA-restricted FVIII epitope with a narrow TCR repertoire.

Factor VIII (FVIII)-neutralizing antibodies ("inhibitors") are a serious problem in hemophilia A (HA). The aim of this study was to characterize HLA-restricted T-cell responses from a severe HA subject with a persistent inhibitor and from 2 previously studied mild HA inhibitor subjects. Major histocompatibility complex II tetramers corresponding to both of the severe HA subject's HLA-DRA-DRB1 alleles were loaded with peptides spanning FVIII-A2, C1, and C2 domains. Interestingly, only 1 epitope was identified, in peptide FVIII2194-2213, and it was identical to the HLA-DRA*01-DRB1*01:01-restricted epitope recognized by the mild HA subjects. Multiple T-cell clones and polyclonal lines having different avidities for the peptide-loaded tetramer were isolated from all subjects. Only high- and medium-avidity T cells proliferated and secreted cytokines when stimulated with FVIII2194-2213 T-cell receptor ß (TCRB) gene sequencing of 15 T-cell clones from the severe HA subject revealed that all high-avidity clones expressed the same TCRB gene. High-throughput immunosequencing of high-, medium-, and low-avidity cells sorted from a severe HA polyclonal line revealed that 94% of the high-avidity cells expressed the same TCRB gene as the high-avidity clones. TCRB sequencing of clones and lines from the mild HA subjects also identified a limited TCRB gene repertoire. These results suggest a limited number of epitopes in FVIII drive inhibitor responses and that the T-cell repertoires of FVIII-responsive T cells can be quite narrow. The limited diversity of both epitopes and TCRB gene usage suggests that targeting of specific epitopes and/or T-cell clones may be a promising approach to achieve tolerance to FVIII.

HCV core expression in hepatocytes protects against autoimmune liver injury and promotes liver regeneration in mice.

Hepatitis C virus (HCV) infection causes acute and chronic liver disease often leading to liver cirrhosis and hepatocellular carcinoma. Numerous studies have shown that despite induction of virus specific immunity, a curative response is often not attained; this has led to the hypothesis that HCV genes modulate immunity, thereby enabling chronic infections. This study examined the effects on immune-mediated liver injury in transgenic mice expressing core protein throughout the body and bone marrow chimeras expressing core protein in either the lymphoid compartment or liver parenchyma. Presence of core protein in the liver parenchyma but not in lymphoid cells protects from autoimmune hepatitis induced by mitogen concanavalin A (ConA). Consistent with this observation, core transgenic hepatocytes are relatively resistant to death induced by anti-Fas antibody and tumor necrosis factor alpha (TNFalpha). This protective effect is associated with preferential activation of signal transducer and activation of transcription factor 3 (STAT3) versus STAT1 in livers of ConA-injected animals. In agreement with this effect of core protein on the Janus kinase (JAK)-STAT signaling pathway, transgenic mice accelerate liver regeneration after partial hepatectomy but are not protected from hepatocyte death. In conclusion, HCV core inhibits STAT1 and stimulates STAT3 activation, which protects infected hepatocytes from attack by the cell-mediated immune system and promotes their proliferation.

P2X7 receptors regulate NKT cells in autoimmune hepatitis.

Adenine nucleotides induce danger signals in T cells via purinergic receptors, raising the question whether they exert similar effects on innate immunity. Here we show that micromolar concentrations of nicotinamide adenine dinucleotide (NAD) induce a rapid increase of annexin V staining in NKT cells in vitro, a response that requires expression of P2X(7)Rs. Consistent with this result, treatment of mice with NAD causes a temporary decrease of NKT cells in the liver and protects from Con A- and alpha-galactosylceramide-induced hepatitis, both of which require functional NKT cells. Resistance to liver injury is associated with decreased cytokine production by NKT cells in NAD-treated mice. In contrast, when NAD is injected into Con A- or alpha-galactosylceramide-primed mice, liver injury is exacerbated and cytokine production by NKT cells is increased. This effect is caused by P2X(7)R-mediated stimulation of activated NKT cells. In agreement, mice lacking P2X(7)Rs on lymphocytes suffer reduced liver injury, and animals lacking ADP-ribosyltransferase, the enzyme that uses NAD to attach ADP-ribosyl groups to cell surfaces, are also resistant to Con A-induced hepatitis. These results prompt the conclusion that engagement of P2X(7)Rs on NKT cells inhibits naive, while stimulating activated cells, resulting in suppression or stimulation of autoimmune hepatitis.

The role of NKT cells in animal models of autoimmune hepatitis.

NKT cells expressing invariant T-cell receptors are an abundant cell population in the mouse liver, and much evidence has been accumulating which shows that they play an important role in immune responses in this organ. In this review, the putative function of NKT cells in autoimmune hepatitis is discussed based on results from various mouse models. Features and functions of invariant NKT cells are summarized to set the stage to explain how these cells induce liver injury following the injection of mitogen concanavalin A or NKT-cell receptor ligand alpha-GalCer. Results are discussed which show that alcohol consumption can aggravate liver injury by NKT cells, whereas expression of a hepatitis C virus protein in hepatocytes can protect against injury. Hepatocytes, therefore, can modulate sensitivity to NKT-cell-mediated attack. Moreover, experiments that elucidate how NKT cells induce liver injury and how they are regulated to perform this function are discussed. Specific attention is given to the recently discovered role of purinergic receptor P2X7 in regulating NKT cells. The conclusion is drawn that the P2X7 receptor constitutes a sensor that senses purine-based danger signals, which trigger mechanisms that cause inhibition or stimulation of NKT-cell functions.

P2X7 receptor expression levels determine lethal effects of a purine based danger signal in T lymphocytes.

Contact of T lymphocytes with nicotinamide adenine dinucleotide (NAD) or ATP causes cell death that requires expression of purinergic receptor P2X(7) (P2X(7)R). T cell subsets differ in their responses to NAD and ATP, which awaits a mechanistic explanation. Here, we show that sensitivity to ATP correlates with P2X(7)R expression levels in CD4 cells, CD8 cells and CD4(+)CD25(+) cells from both C57BL/6 and BALB/c mice. But P2X(7)R ligands do not only induce cell death but also shedding of CD62L. It is shown here that in CD62L(high) T cells, CD62L shedding correlates with low expression of P2X(7)Rs and lower cell death, whereas in CD62L(low) cells P2X(7)R expression and death are higher. The possibility is therefore investigated that P2X(7)Rs induce T cell activation. Experiments show that spontaneous T cell proliferation is somewhat higher in cells expressing P2X(7)Rs, but this effect we suggest is caused by P2X(7)R expression on accessory cells.

High sensitivity of CD4+CD25+ regulatory T cells to extracellular metabolites nicotinamide adenine dinucleotide and ATP: a role for P2X7 receptors.

Although regulatory lymphocytes play an important role in the immune system, the regulation of their functions is poorly understood and remains to be elucidated. In this study we demonstrate that micromolar concentrations of the common cell metabolite NAD induce death in murine forkhead/winged helix transcription factor gene-expressing CD4+CD25+ regulatory T cells with high efficiency and within minutes. Similar, but less dramatic, effects are demonstrable with ATP and its nonhydrolysable derivative, benzoylbenzoyl-ATP. Other T cell subsets are more resistant, with CD8 cells being the least sensitive and CD4 cells expressing intermediate sensitivity. The higher sensitivity of CD4+CD25+ cells is demonstrable in vivo. Injection of NAD or benzoylbenzoyl-ATP causes preferential induction of a cell death signal in CD4+CD25+ cells. Transmission of the death signal requires functional P2X7 receptors, pointing to a role for these receptors in regulation and homeostasis of CD4+CD25+ regulatory T cells. Consistent with this, P2X7R gene-deleted mice possess increased levels of forkhead/winged helix transcription factor gene-expressing CD4+CD25+ cells.

P2X7 receptor-dependent and -independent T cell death is induced by nicotinamide adenine dinucleotide.

Adding NAD to murine T lymphocytes inhibits their functions and induces annexin V binding. This report shows that NAD induces cell death in a subset of T cells within seconds whereas others do not die until many hours later. Low NAD concentrations (<10 microM) suffice to trigger rapid cell death, which is associated with annexin V binding and membrane pore formation, is not blocked by the caspase inhibitor Z-VADfmk, and requires functional P2X7 receptors. The slower induction of death requires higher NAD concentrations (>100 microM), is blocked by caspase inhibitor Z-VADfmk, is associated with DNA fragmentation, and does not require P2X7 receptors. T cells degrade NAD to ADP-ribose (ADPR), and adding ADPR to T cells leads to slow but not rapid cell death. NAD but not ADPR provides the substrate for ADP-ribosyltransferase (ART-2)-mediated attachment of ADP-ribosyl groups to cell surface proteins; expression of ART-2 is required for NAD to trigger rapid but not slow cell death. These results support the hypothesis that cell surface ART-2 uses NAD but not ADPR to attach ADP-ribosyl groups to the cell surface, and that these groups act as ligands for P2X7 receptors that then induce rapid cell death. Adding either NAD or ADPR also triggers a different set of mechanisms, not requiring ART-2 or P2X7 receptors that more slowly induce cell death.