ALBERT

Description: Key PointsCD4+CD25+FoxP3+ T regulatory cells and CD11c+ dendritic cells protect against antibody-mediated murine TRALI. Murine TRALI is associated with reduced IL-10 levels, and IL-10 administration prevents and rescues TRALI development.

Description: Transfusion-related acute lung injury (TRALI) is one of the leading causes of transfusion-related fatalities and is characterized by the onset of acute respiratory distress within 6 hours upon blood transfusion. Specific therapies are unavailable. Preexisting inflammation is a risk factor for TRALI and neutrophils (polymorphonuclear neutrophils [PMNs]) are considered to be the major pathogenic cells. Osteopontin (OPN) is a multifunctional protein expressed at sites of inflammation and, for example, is involved in pulmonary disorders, can regulate cellular migration, and can function as a PMN chemoattractant. We investigated whether OPN is involved in TRALI induction by promoting PMN recruitment to the lungs. Using a previously established murine TRALI model, we found that in contrast to wild-type (WT) mice, OPN knockout (KO) mice were resistant to antibody-mediated PMN-dependent TRALI induction. Administration of purified OPN to the OPN KO mice, however, restored the TRALI response and pulmonary PMN accumulation. Alternatively, blockade of OPN in WT mice using an anti-OPN antibody prevented the onset of TRALI induction. Using pulmonary immunohistochemistry, OPN could be specifically detected in the lungs of mice that suffered from TRALI. The OPN-mediated TRALI response seemed dependent on macrophages, likely the cellular source of OPN and OPN polymerization, and independent from the OPN receptor CD44, interleukin 6 (IL-6), and other PMN chemoattractants including macrophage inflammatory protein-2 (MIP-2). These data indicate that OPN is critically required for induction of antibody-mediated murine TRALI through localization to the lungs and stimulation of pulmonary PMN recruitment. This suggests that anti-OPN antibody therapy may be a potential therapeutic strategy to explore in TRALI patients.

Description: Transfusion-associated circulatory overload (TACO) and transfusion-related acute lung injury (TRALI) are syndromes of acute respiratory distress that occur within 6 hours of blood transfusion. TACO and TRALI are the leading causes of transfusion-related fatalities, and specific therapies are unavailable. Diagnostically, it remains very challenging to distinguish TACO and TRALI from underlying causes of lung injury and/or fluid overload as well as from each other. TACO is characterized by pulmonary hydrostatic (cardiogenic) edema, whereas TRALI presents as pulmonary permeability edema (noncardiogenic). The pathophysiology of both syndromes is complex and incompletely understood. A 2-hit model is generally assumed to underlie TACO and TRALI disease pathology, where the first hit represents the clinical condition of the patient and the second hit is conveyed by the transfusion product. In TACO, cardiac or renal impairment and positive fluid balance appear first hits, whereas suboptimal fluid management or other components in the transfused product may enable the second hit. Remarkably, other factors beyond volume play a role in TACO. In TRALI, the first hit can, for example, be represented by inflammation, whereas the second hit is assumed to be caused by antileukocyte antibodies or biological response modifiers (eg, lipids). In this review, we provide an up-to-date overview of TACO and TRALI regarding clinical definitions, diagnostic strategies, pathophysiological mechanisms, and potential therapies. More research is required to better understand TACO and TRALI pathophysiology, and more biomarker studies are warranted. Collectively, this may result in improved diagnostics and development of therapeutic approaches for these life-threatening transfusion reactions.

Description: Key Points CD20 Bdep therapy inhibits CD8+ T-cell proliferation in vitro. CD20 Bdep therapy prevents CD8+ T-cell–mediated ITP in vivo.

Description: Key Points CRP enhances antibody-mediated lung damage when infused into TRALI-resistant mice. CRP and TRALI-inducing antibodies generate a synergistic increase in MIP-2 production and pulmonary neutrophil accumulation in vivo.

Description: Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by low platelet counts. ITP has a complex pathogenesis, in which both anti-platelet antibodies as well as T cells have been shown to be important. Initial management of newly diagnosed ITP may be either watchful waiting or pharmacologic intervention, such as glucocorticoids or Intravenous Immunoglobulin (IVIg), a blood product consisting of polyclonal immunoglobulin G (IgG) derived from thousands of donors. Second-line therapy may include dexamethasone, high-dose methylprednisolone, rituximab, thrombopoietin (TPO)-receptor agonists, or splenectomy. The working mechanism of IVIg is actively under investigation and is still a matter of debate, as various different working mechanisms have been suggested. One of them is that IVIg may shift the balance from a pro- to anti-inflammatory state through immunomodulating the activity of dendritic cells (DCs). To gain more insights into the role of DCs in ITP, upon IVIg treatment or splenectomy, we analyzed DC subsets in a murine model of ITP, which features both the antibody and T cell mediated thrombocytopenia. Severe combined immunodeficient (SCID) mice were administrated 4x104 splenocytes from CD61 (GPIIIa) knockout mice immunized against CD61 (or naïve control splenocytes) and the mice were treated with or without 1 g/kg IVIg twice a week. Also the same type of splenocytes were transferred into splenectomized SCID mice. Weekly platelet counts were assessed and after 4 weeks the mice were sacrificed and spleen and thymuses were harvested. Splenocytes and thymocytes were isolated and examined by flow cytometry for cross-presenting (XCR1+) and non-presenting tolerizing (SIRP alpha+) DCs. Without IVIg or splenectomy, both splenic DC subset numbers correlated positively with platelet counts and both the thymic DC subset numbers correlated negatively with platelet counts, indicating thymic retention of DC in a setting of thrombocytopenia. Interestingly, splenectomized SCID mice, apart from increased platelet counts, demonstrated a complete reversal of the DC pattern in the thymus, as thymic DC subsets correlated positively with platelet counts in splenectomized mice. Upon IVIg treatment, apart from a general increase in platelet counts, the splenic tolerizing DCs significantly increased in numbers. Moreover, the thymic retention of tolerizing DCs and thus the negative correlation with platelet counts (R2: 0.46, p

Description: Major Histocompatibility complex (MHC) class I is a complex composed of two proteins, the MHC-encoded alpha heavy chain and the beta2-microglobulin light chain. In the endoplasmic reticulum, it receives an antigen peptide of usually eight to eleven residues in its groove and is exported to the plasma membrane. In classical MHC class I presentation, endogenous antigens are presented to CD8+ T cells. On the other hand, cross-presentation consists in the presentation of exogenously-derived proteins in the context of MHC class I molecules to CD8+ T lymphocytes, inducing a cytotoxic response against the antigen presenting cell (APC). Platelets have been previously shown to be able to cross-present immunogenic ovalbumin (OVA) peptides, as well as bacterial peptides and to trigger CD8+ T lymphocyte activation. It is thought that the antigen cross presenting function of platelets is transmitted from megakaryocytes (MK) during thrombopoiesis, but whether megakaryocytes can cross-present is still unknown. To address this question, we developed an in vitro endocytosis model of murine bone marrow-derived megakaryocytes. C57BL/6 mouse megakaryocytes are enriched by gradient sedimentation and pulsed with fluorescent ovalbumin (OVA) for various times. In addition, we used DQ-OVA, a BODIPY FL-conjugated OVA, which becomes fluorescent upon proteolysis. Cells were then analyzed by flow cytometry or confocal microscopy to assess the OVA megakaryocyte internalization, its proteolytic degradation and its loading onto MHC class I molecules. We can show the endocytosis of exogenous OVA by megakaryocytes, in a pathway involving lysosomes. The endocytosed OVA were subsequently proteolytically cleaved into the immunogenic OVA peptide SIINFEKL and the peptide is inserted into the antigen binding groove of MHC class I molecules. The MHC class I-OVA peptide complexes are stored in the MK alpha-granules and can be translocated to the plasma membrane. Thus, we have shown that MKs can effectively process and present exogenous antigens on MHC class I molecules. This opens a new avenue toward a functional characterization of this pathway and its modulation, leading to new therapeutic approaches in autoimmune diseases targeting megakaryocytes, such as immune thrombocytopenia (ITP). Disclosures No relevant conflicts of interest to declare.

Description: Transfusion related acute lung injury (TRALI) is the leading cause of transfusion-induced fatalities and is characterized by acute respiratory distress following blood transfusion. Donor antibodies present in the transfused blood product such as anti-human leukocyte antigen (HLA) or anti-human neutrophil antigen (HNA) antibodies are frequently involved. Currently, there is no treatment available for TRALI apart from supportive measures such as oxygen. The pathogenesis the disorder is incompletely understood, however, several animal models have contributed to our understanding of TRALI disease pathology. Most TRALI reactions are considered to be due to a two-hit paradigm where the first hit is a predisposing patient factor such as inflammation while the second hit is the transfusion. It is widely believed that the second hit delivers antibodies that trigger TRALI in the recipient. The anti-MHC class I antibody, 34-1-2s, has been widely used as an agent that delivers the second TRALI hit in mice. We have previously shown that CD4+ T cells, more specifically, CD4+CD25+FoxP3+ T-regulatory cells (Tregs) convey protection against TRALI (Blood. 126 (23):2342, 2015; abstract #82075, manuscript submitted). In the current study, we utilized a C57BL/6 mouse model of severe TRALI by first depleting mice of CD4+ T cells and then injecting them with the anti-MHC class I monoclonal antibodies (34-1-2s+AF6-88.5.5.3) and we examined the effects of the anti-inflammatory cytokine IL-10 on the antibody-mediated TRALI reaction. IL-10 (45 µg/kg iv) or volume-matched PBS was injected 15 minutes after the administration of anti-MHC antibodies when the onset of TRALI symptoms (e.g. a 2 degree drop in rectal temperature indicative of systemic shock) began. Results show that 90 minutes after anti-MHC class I antibody injection, control mice injected with PBS exhibited a high degree of pulmonary edema as assessed by significantly elevated lung wet-to-dry weight ratios (W/D: 5.84 ± 1.02). Pulmonary neutrophil levels were also found to be increased and lung tissue histology confirmed severe signs of acute lung injury. In contrast, mice injected with IL-10 completely recovered from TRALI; after 90 minutes post-antibody injection they displayed no signs of pulmonary edema (W/D: 4.76 ± 0.04, ** p

Description: Transfusion-related acute lung injury (TRALI) is a syndrome of respiratory distress which develops within 6 hours of blood transfusion. It is the leading cause of transfusion-related deaths and the pathogenesis is complex and incompletely understood. In the majority of the cases, anti-leukocyte antibodies present in the transfused blood product, in combination with recipient predisposing risk-factors such as inflammation, are implicated to be responsible for the onset of TRALI. Unfortunately, no therapies are available for TRALI. Osteopontin (OPN) is an extracellular matrix protein with multiple biological functions. OPN is involved in normal physiological processes, such as cell migration and adhesion, but has also been implicated in a wide range of disease states, including cancer, atherosclerosis, glomerulonephritis, and several chronic inflammatory diseases. Interestingly, OPN is upregulated at sites of inflammation and tissue remodeling. As inflammation is an important risk factor for TRALI development, and as neutrophils (PMNs) are known effector cells in the pathogenesis of TRALI which migrate and accumulate in the lungs during TRALI development, we investigated the potential contribution of OPN in the onset of antibody-mediated TRALI. We utilized a previously established murine TRALI model (Kapur et al, Blood 2017, Blood Advances 2018) in which C57BL/6 mice were first primed with a low dose of lipopolysaccharide (LPS) and depleted of their CD4+ T cells in vivo followed by injection of anti-major histocompatibility complex (MHC) class I antibodies (clones 34-1-2s and AF6-88.5.5.3). The TRALI response was analyzed after 90 minutes by analysis of pulmonary edema (lung wet-to-dry weight ratios, W/Ds) and the levels of pulmonary neutrophils. Wildtype (WT) mice suffered from antibody-mediated TRALI compared to untreated naïve mice, as was shown by their significantly increased lung W/Ds (4.72 vs 4.50, respectively, P

Description: Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder in which autoantibodies and autoreactive T cells target the destruction of platelets and megakaryocytes in the spleen and bone marrow. Several therapeutic options e.g. steroids, IVIg, Rituximab and thrombopoietin mimetics, are available for patients but inadequate efficacy, side effects and/or expense can make them undesirable. PRTX-100 is a highly purified formulation of Staphylococcal Protein A that has been evaluated in clinical trials with rheumatoid arthritis patients. Here, we analyzed the efficacy of PRTX-100 in raising platelet counts in a well-established murine model of ITP that demonstrates both antibody- and T cell-mediated thrombocytopenia. Platelet glycoprotein (GP) IIIa (CD61) knockout (KO) mice were immunized with CD61+ platelets and ITP was initiated by transfer of their splenocytes into severe combined immunodeficient (SCID) mice. On day 10, post transfer, the SCID mice were treated with either placebo, 1g/kg IVIg (ip, biweekly) or doses of PRTX-100 (iv, biweekly, 2.5-250 ug/kg) and platelet counts were measured weekly. Results show that control SCID mice transferred with 1x104 splenocytes from immune CD61 KO mice became thrombocytopenic at day 7 post-transfer and remained extremely thrombocytopenic throughout the 28 day protocol (3 mice died from bleeding diatheses). Similar observations were found with SCID mice given placebo. In contrast, however, transferred SCID mice treated with either IVIg or the PRTX-100 doses had platelet counts that increased to within normal levels within 1-2 weeks after treatment and none of the mice died (Table 1). Table 1.Platelet counts (mean+SD) in SCID ITP mice (N=3-8/group) treated with the indicated compounds.TreatmentPre-bleedDay 14Day 21Day 28No treatment848+20289+4651+4278+51Placebo (250 ug/kg)848+202113+5946+2063+40IVIg (1 g/kg)848+202290+211490+289698+350PRTX-100 (250 ug.kg)848+202101+37303+112505+226PRTX-100 (25 ug.kg)848+202203+88290+67254+70PRTX-100 (2.5 ug.kg)848+202275+202405+289631+401 These results demonstrate that PRTX-100 was effective in elevating platelet counts in a murine model of human ITP and support the proof of principle that PRTX-100 may be beneficial in patients with ITP. Disclosures No relevant conflicts of interest to declare.