While it is known that CD123 is normally strongly expressed on plasmacytoid dendritic cells and completely absent on nucleated red blood cells, detailed information regarding CD123 expression in acute leukemia is scarce and, if available, hard to compare due to different methodologies.

Activation of the classical and lectin pathway of complement may contribute to tissue damage and organ dysfunction of antibody-mediated diseases and ischemia-reperfusion conditions. Complement factors are being considered as targets for therapeutic intervention.

Dendritic cells (DCs) are efficient antigen-presenting cells equipped with various cell surface receptors for the direct or indirect recognition of pathogenic microorganisms. Interestingly, not much is known about the specific expression pattern and function of the individual activating and inhibitory Fcγ receptors (FcγRs) on splenic DC subsets in vivo and how they contribute to the initiation of T cell responses. By targeting antigens to select activating and the inhibitory FcγR in vivo, we show that antigen uptake under steady-state conditions results in a short-term expansion of antigen-specific T cells, whereas under inflammatory conditions especially, the activating FcγRIV is able to induce superior CD4+ and CD8+ T cell responses. Of note, this effect was independent of FcγR intrinsic activating signaling pathways. Moreover, despite the expression of FcγRIV on both conventional splenic DC subsets, the induction of CD8+ T cell responses was largely dependent on CD11c+CD8+ DCs, whereas CD11c+CD8- DCs were critical for priming CD4+ T cell responses.

Immunotherapy targeting GD2 is very effective against high-risk neuroblastoma, though administration of anti-GD2 antibodies induces severe and dose-limiting neuropathic pain by binding GD2-expressing sensory neurons. Previously, the IgG1 ch14.18 (dinutuximab) antibody was reformatted into the IgA1 isotype, which abolishes neuropathic pain and induces efficient neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) via activation of the Fc alpha receptor (FcαRI/CD89).

Neutrophils are crucial innate immune cells but also play key roles in various diseases, such as cancer, where they can perform both pro- and anti-tumorigenic functions. To study the function of neutrophils in vivo, these cells are often depleted using Ly-6G or Gr-1 depleting antibodies or genetic "knockout" models. However, these methods have several limitations, being only partially effective, effective for a short term, and lacking specificity or the ability to conditionally deplete neutrophils. Here, we describe the use of a novel murinized Ly-6G (1A8) antibody. The murinized Ly-6G antibody is of the mouse IgG2a isotype, which is the only isotype that can bind all murine Fcγ receptors and C1q and is, therefore, able to activate antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) pathways. We show that this mouse-Ly-6G antibody shows efficient, long-term, and near-complete (>90%) neutrophil depletion in the peripheral blood of C57Bl6/J, Balb/c, NXG and SCID mice for up to at least four weeks, using a standardized neutrophil depletion strategy. In addition, we show that neutrophils are efficiently depleted in the blood and tumor tissue of IMR32 tumor-bearing SCID mice, analyzed six weeks after the start of the treatment.

New therapeutic approaches to resolve persistent pain are highly needed. We tested the hypothesis that manipulation of cytokine receptors on sensory neurons by clustering regulatory cytokine receptor pairs with a fusion protein of interleukin (IL)-4 and IL-10 (IL4-10 FP) would redirect signaling pathways to optimally boost pain-resolution pathways. We demonstrate that a population of mouse sensory neurons express both receptors for the regulatory cytokines IL-4 and IL-10. This population increases during persistent inflammatory pain. Triggering these receptors with IL4-10 FP has unheralded biological effects, because it resolves inflammatory pain in both male and female mice. Knockdown of both IL4 and IL10 receptors in sensory neurons in vivo ablated the IL4-10 FP-mediated inhibition of inflammatory pain. Knockdown of either one of the receptors prevented the analgesic gain-of-function of IL4-10 FP. In vitro, IL4-10 FP inhibited inflammatory mediator-induced neuronal sensitization more effectively than the combination of cytokines, confirming its superior activity. The IL4-10 FP, contrary to the combination of IL-4 and IL-10, promoted clustering of IL-4 and IL-10 receptors in sensory neurons, leading to unique signaling, that is exemplified by activation of shifts in the cellular kinome and transcriptome. Interrogation of the potentially involved signal pathways led us to identify JAK1 as a key downstream signaling element that mediates the superior analgesic effects of IL4-10 FP. Thus, IL4-10 FP constitutes an immune-biologic that clusters regulatory cytokine receptors in sensory neurons to transduce unique signaling pathways required for full resolution of persistent inflammatory pain.

Collagen expression and structure in the tumour microenvironment are associated with tumour development and therapy response. Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is a widely expressed inhibitory collagen receptor. LAIR-2 is a soluble homologue of LAIR-1 that competes for collagen binding. Multiple studies in mice implicate blockade of LAIR-1:collagen interaction in cancer as a promising therapeutic strategy. Here, we investigated the role of LAIR-1 in anti-tumour responses. We show that although LAIR-1 inhibits activation, proliferation, and cytokine production of mouse T cells in vitro, tumour outgrowth in LAIR-1-deficient mice did not differ from wild type mice in several in vivo tumour models. Furthermore, treatment with NC410, a LAIR-2-Fc fusion protein, did not result in increased tumour clearance in tested immunocompetent mice, which contrasts with previous data in humanized mouse models. This discrepancy may be explained by our finding that NC410 blocks human LAIR-1:collagen interaction more effectively than mouse LAIR-1:collagen interaction. Despite the lack of therapeutic impact of NC410 monotherapy, mice treated with a combination of NC410 and anti-programmed death-ligand 1 did show reduced tumour burden and increased survival. Using LAIR-1-deficient mice, we showed that this effect seemed to be dependent on the presence of LAIR-1. Taken together, our data demonstrate that the absence of LAIR-1 signalling alone is not sufficient to control tumour growth in multiple immunocompetent mouse models. However, combined targeting of LAIR-1 and PD-L1 results in increased tumour control. Thus, additional targeting of the LAIR-1:collagen pathway with NC410 is a promising approach to treating tumours where conventional immunotherapy is ineffective.

Obstructive chronic lung allograft dysfunction (BOS) is the major limiting factor for lung transplantation (LTx) outcome. PTPN22 is described as the hallmark autoimmunity gene, and one specific single nucleotide polymorphism (SNP), rs2476601, is associated with multiple autoimmune diseases, impaired T cell regulation, and autoantibody formation. Taking into consideration the contribution of autoimmunity to LTx outcome, we hypothesized that polymorphisms in the PTPN22 gene could be associated with BOS incidence. We selected six SNPs within PTPN22 and analyzed both patient and donor genotypes on BOS development post-LTx. A total of 144 patients and matched donors were included, and individual SNPs and haplotype configurations were analyzed. We found a significant association between patients carrying the heterozygous configuration of rs2476601 and a higher risk for BOS development (p = 0.005, OR: 4.400, 95%CI: 1.563-12.390). Kaplan-Meier analysis showed that heterozygous patients exhibit a lower BOS-free survival compared to patients homozygous for rs2476601 (p = 0.0047). One haplotype, which solely contained the heterozygous risk variant, was associated with BOS development (p = 0.015, OR: 7.029, 95%CI: 1.352-36.543). Our results show that LTx patients heterozygous for rs2476601 are more susceptible for BOS development and indicate a deleterious effect of the autoimmune-related risk factor of PTPN22 in patients on LTx outcome.

Clinical trials with SRC family kinases (SFKs) inhibitors used alone or in a combination with anti-CD20 monoclonal antibodies (mAbs) are currently underway in the treatment of B-cell tumors. However, molecular interactions between these therapeutics have not been studied so far. A transcriptional profiling of tumor cells incubated with SFKs inhibitors revealed strong downregulation of MS4A1 gene encoding CD20 antigen. In a panel of primary and established B-cell tumors we observed that SFKs inhibitors strongly affect CD20 expression at the transcriptional level, leading to inhibition of anti-CD20 mAbs binding and increased resistance of tumor cells to complement-dependent cytotoxicity. Activation of the AKT signaling pathway significantly protected cells from dasatinib-triggered CD20 downregulation. Additionally, SFKs inhibitors suppressed antibody-dependent cell-mediated cytotoxicity by direct inhibition of natural killer cells. Abrogation of antitumor activity of rituximab was also observed in vivo in a mouse model. Noteworthy, the effects of SFKs inhibitors on NK cell function are largely reversible. The results of our studies indicate that development of optimal combinations of novel treatment modalities with anti-CD20 mAbs should be preceded by detailed preclinical evaluation of their effects on target cells.

Pro-survival BCL-2 family members protect cells from programmed cell death that can be induced by multiple internal or external cues. Within the haematopoietic lineages, the BCL-2 family members BCL-2, BCL-XL and MCL-1 are known to support cell survival but the individual and overlapping roles of these pro-survival BCL-2 proteins for the persistence of individual leukocyte subsets in vivo has not yet been determined. By combining inducible knockout mouse models with the BH3-mimetic compound ABT-737, which inhibits BCL-2, BCL-XL and BCL-W, we found that dependency on MCL-1, BCL-XL or BCL-2 expression changes during B-cell development. We show that BCL-XL expression promotes survival of immature B cells, expression of BCL-2 is important for survival of mature B cells and long-lived plasma cells (PC), and expression of MCL-1 is important for survival throughout B-cell development. These data were confirmed with novel highly specific BH3-mimetic compounds that target either BCL-2, BCL-XL or MCL-1. In addition, we observed that combined inhibition of these pro-survival proteins acts in concert to delete specific B-cell subsets. Reduced expression of MCL-1 further sensitized immature as well as transitional B cells and splenic PC to loss of BCL-XL expression. More markedly, loss of MCL-1 greatly sensitizes PC populations to BCL-2 inhibition using ABT-737, even though the total wild-type PC pool in the spleen is not significantly affected by this drug and the bone marrow (BM) PC population only slightly. Combined loss or inhibition of MCL-1 and BCL-2 reduced the numbers of established PC >100-fold within days. Our data suggest that combination treatment targeting these pro-survival proteins could be advantageous for treatment of antibody-mediated autoimmune diseases and B-cell malignancies.

Respiratory syncytial virus (RSV) infection is a leading cause of hospitalization and mortality in young children. Protective therapy options are limited. Currently, palivizumab, a monoclonal IgG1 antibody, is the only licensed drug for RSV prophylaxis, although other IgG antibody candidates are being evaluated. However, at the respiratory mucosa, IgA antibodies are most abundant and act as the first line of defense against invading pathogens. Therefore, it would be logical to explore the potential of recombinant human IgA antibodies to protect against viral respiratory infection, but very little research on the topic has been published. Moreover, it is unknown whether human antibodies of the IgA isotype are better suited than those of the IgG isotype as antiviral drugs to combat respiratory infections. To address this, we generated various human IgA antibody formats of palivizumab and motavizumab, two well-characterized human IgG1 anti-RSV antibodies. We evaluated their efficacy to prevent RSV infection in vitro and in vivo and found similar, but somewhat decreased efficacy for different IgA subclasses and formats. Thus, reformatting palivizumab or motavizumab into IgA reduces the antiviral potency of either antibody. Moreover, our results indicate that the efficacy of intranasal IgA prophylaxis against RSV infection in human FcαRI transgenic mice is independent of Fc receptor expression.

Neuroblastoma is one of the most commonly found solid tumors in children. The monoclonal antibody dinutuximab (DNX) targets the sialic acid-containing glycosphingolipid GD2 expressed on almost all neuroblastoma tumor cells and induces cell lysis. However, the expression of GD2 is not limited to tumor cells only, but is also present on central nerve tissue and peripheral nerve cells explaining dinutuximab toxicity. The most common adverse reactions are pain and discomfort, which may lead to discontinuation of the treatment. Furthermore, there is little to no data available on exposure and effect relationships of dinutuximab. We, therefore, developed an easy method in order to quantify dinutuximab levels in human plasma. Ammonium sulfate (AS) was used to precipitate all immunoglobulins (IgGs) in human plasma. After centrifugation, supernatant containing albumin was decanted and the precipitated IgG fraction was re-dissolved in a buffer containing 0.5% sodium dodecyl sulfate (SDS). Samples were then reduced, alkylated, and digested with trypsin. Finally, a signature peptide in complementarity determining region 1 of DNX heavy chain was quantified on LC-MS/MS using a stable isotopically labeled peptide as internal standard. AS purification efficiently removed 97.5% of the albumin fraction in the supernatant layer. The validation performed on DNX showed that within-run and between-run coefficients of variation (CV) for lower limit of quantification (LLOQ) were 5.5 and 1.4%, respectively. The overall CVs for quality control (QC) low, QC med, and QC high levels were < 5%. Linearity in the range 1-32 mg/L was excellent (r2 > 0.999). Selectivity, stability, and matrix effect were in concordance with EMA guidelines. In conclusion, a method to quantify DNX in human plasma was successfully developed. In addition, the high and robust process efficiency enabled the utilization of a stable isotopically labeled (SIL) peptide instead of SIL DNX, which was commercially unavailable. Graphical abstract.

Transplacental respiratory syncytial virus (RSV) antibody transfer has been characterized, but little is known about the protective effect of breast milk RSV-specific antibodies. Serum antibodies against the prefusion RSV fusion protein (pre-F) exhibit high neutralizing activity. We investigate protection of breast milk pre-F antibodies against RSV acute respiratory infection (ARI).

Currently all approved anti-cancer therapeutic monoclonal antibodies (mAbs) are of the IgG isotype, which rely on Fcgamma receptors (FcγRs) to recruit cellular effector functions. In vitro studies showed that targeting of FcαRI (CD89) by bispecific antibodies (bsAbs) or recombinant IgA resulted in more effective elimination of tumour cells by myeloid effector cells than targeting of FcγR. Here we studied the in vivo anti-tumour activity of IgA EGFR antibodies generated using the variable sequences of the chimeric EGFR antibody cetuximab. Using FcαRI transgenic mice, we demonstrated significant in vivo anti-tumour activity of IgA2 EGFR against A431 cells in peritoneal and lung xenograft models, as well as against B16F10-EGFR cells in a lung metastasis model in immunocompetent mice. IgA2 EGFR was more effective than cetuximab in a short-term syngeneic peritoneal model using EGFR-transfected Ba/F3 target cells. The in vivo cytotoxic activity of IgA2 EGFR was mediated by macrophages and was significantly decreased in the absence of FcαRI. These results support the potential of targeting FcαRI for effective antibody therapy of cancer.

Subsequent to myocardial infarction, cardiomyocytes within the infarcted areas and border zones expose phosphatidylserine (PS) in the outer plasma membrane leaflet (flip-flop). We showed earlier that in addition to apoptosis, this flip-flop can be reversible in cardiomyocytes. We now investigated a possible role for Rho and downstream effector Rho-associated kinase (ROCK) in the process of (reversible) PS exposure and apoptosis in cardiomyocytes. In rat cardiomyoblasts (H9c2 cells) and isolated adult ventricular rat cardiomyocytes Clostridium difficile Toxin B (TcdB), a Rho GTPase family inhibitor, C3 transferase (C3), a Rho(A,B,C) inhibitor and the ROCK inhibitors Y27632 and H1152 were used to inhibit Rho-ROCK signaling. PS exposure was assessed via flow cytometry and fluorescent digital imaging microscopy using annexin V. Akt expression and phosphorylation were analyzed via Western blot, and Akt activity was inhibited by wortmannin. The cellular concentration activated caspase 3 was determined as a measure of apoptosis, and flippase activity was assessed via flow cytometry using NBD-labeled PS. TcdB, C3, Y27632 and H1152 all significantly increased PS exposure. TcdB, Y27632 and H1152 all significantly inhibited phosphorylation of the anti-apoptotic protein Akt and Akt inhibition by wortmannin lead to increased PS exposure. However, only TcdB and C3, but not ROCK- or Akt inhibition led to caspase 3 activation and thus apoptosis. Notably, pancaspase inhibitor zVAD only partially inhibited TcdB-induced PS exposure indicating the existence of apoptotic and non-apoptotic PS exposure. The induced PS exposure coincided with decreased flippase activity as measured with NBD-labeled PS flip-flop. In this study, we show a regulatory role for a novel signaling route, Rho-ROCK-flippase signaling, in maintaining asymmetrical membrane phospholipid distribution in cardiomyocytes.

Antibodies against HLA and non-HLA are associated with transplantation outcome. Recently, pretransplant serum IgG antibody levels against apoptotic cells were found to correlate with kidney allograft loss. We investigated the presence of these antibodies in lung transplantation (LTx) patients and evaluated the correlation of pre-LTx serum levels of IgG antibodies against apoptotic cells with LTx outcome. These cells included donor lung endothelial cells (ECs) obtained from lung perfusion fluid collected during LTx procedure. Cells were isolated, expanded in vitro, and analyzed as targets for antiapoptotic cell reactivity. Cultured cells exhibited EC morphology and were CD31+, CD13+, and vWF+. End-stage lung disease patients showed elevated serum IgG levels against apoptotic lung EC (p = 0.0018) compared to healthy controls. Interestingly, the levels of circulating antibodies directed against either apoptotic Jurkat cells or apoptotic lung ECs did not correlate, suggesting a target cell specificity. We observed no correlation between chronic or acute rejection and pre-LTx serum levels of antiapoptotic antibodies. Also, these levels did not differ between matched patients developing chronic rejection or not during follow-up or at the time of diagnosis, as they remained as high as prior to transplantation. Thus, circulating levels of antiapoptotic cell antibodies are elevated in end-stage lung disease patients, but our data do not correlate with outcome after LTx.

Respiratory syncytial virus (RSV) infections represent a major burden of disease in infants and are the second most prevalent cause of death worldwide. Human milk immunoglobulins provide protection against RSV. However, many infants depend on processed bovine milk-based nutrition, which lacks intact immunoglobulins. We investigated the potential of bovine antibodies to neutralize human RSV and facilitate-cell immune activation. We show cow's milk IgG (bIgG) and Intravenous Immunoglobulin (IVIG) have a similar RSV neutralization capacity, even though bIgG has a lower pre-F to post-F binding ratio compared to human IVIG, with the majority of bIgG binding to pre-F. RSV is better neutralized with human IVIG. Consequently, we enriched RSV specific T cells by culturing human PBMC with a mixture of RSV peptides, and used these T cells to study the effect of bIgG and IVIG on the activation of pre-F-pecific T cells. bIgG facilitated in vitro T cell activation in a similar manner as IVIG. Moreover, bIgG was able to mediate T cell activation and internalization of pathogens, which are prerequisites for inducing an adaptive viral response. Using in vivo mouse experiments, we showed that bIgG is able to bind the murine activating IgG Fc Receptors (FcγR), but not the inhibiting FcγRII. Intranasal administration of the monoclonal antibody palivizumab, but also of bIgG and IVIG prevented RSV infection in mice. The concentration of bIgG needed to prevent infection was ~5-fold higher compared to IVIG. In conclusion, the data presented here indicate that functionally active bIgG facilitates adaptive antiviral T cell responses and prevents RSV infection in vitro and in vivo.

The addition of rabbit anti-human thymocyte globulin (ATG) to the conditioning regimen prior to allogeneic hematopoietic cell transplantation has significantly reduced the risk of graft-versus-host disease (GvHD) and graft failure. However, ATG has a small therapeutic window. Overexposure of ATG post-HCT hampers T cell immune reconstitution and has been associated with increased relapse rates and viral reactivations, whereas underexposure has been associated with an increased incidence of GvHD, both of which lead to increased mortality. Therapeutic drug monitoring of T cell binding ATG plasma levels provides a means to optimize dosing for patients at high risk for graft failure to ensure timely T cell immune reconstitution and subsequently increase survival chances. This manuscript describes the first liquid chromatography tandem-mass spectrometry (LC-MS/MS) method to quantify the pharmacologically active fraction of polyclonal ATG in plasma. This was achieved through immunoaffinity purification of active ATG from plasma with Jurkat T cells. After the binding and washing, samples were eluted, denatured, and trypsin-digested. Signature peptides originating from the IgG constant chain were measured with LC-MS/MS. Critical method parameters were optimized, and the method was successfully validated following European Medicines Agency (EMA) guidelines. The method covered the therapeutic range of ATG and was validated at a lower limit of quantification (LLOQ) of 1 AU/mL with an overall CV and bias of 11.8% and - 2.5%, respectively. In conclusion, we developed a LC-MS/MS-based method to quantify active polyclonal rabbit ATG in human plasma. We suggest that this novel assay can be used to monitor and optimize dosing of ATG in clinical practice.

Patients affected by poverty-related infectious diseases (PRDs) are disproportionally affected by malnutrition. To optimize treatment of patients affected by PRDs, we aimed to assess the influence of malnutrition associated with PRDs on drug pharmacokinetics, by way of a systematic review.

Patients with rare cancers (incidence less than 6 cases per 100,000 persons per year) commonly have less treatment opportunities and are understudied at the level of genomic targets. We hypothesized that patients with rare cancer benefit from approved anticancer drugs outside their label similar to common cancers.