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To monitor infection by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and successful vaccination against coronavirus disease 2019 (COVID‐19), the kinetics of neutralizing or blocking anti‐SARS‐CoV‐2 antibody titers need to be assessed. Here, we report the development of a quick and inexpensive surrogate SARS‐CoV‐2 blocking assay (SUBA) using immobilized recombinant human angiotensin‐converting enzyme 2 (hACE2) and human cells expressing the native form of surface SARS‐CoV‐2 spike protein. Spike protein‐expressing cells bound to hACE2 in the absence or presence of blocking antibodies were quantified by measuring the optical density of cell‐associated crystal violet in a spectrophotometer. The advantages are that SUBA is a fast and inexpensive assay, which does not require biosafety level 2‐ or 3‐approved laboratories. Most importantly, SUBA detects blocking antibodies against the native trimeric cell‐bound SARS‐CoV‐2 spike protein and can be rapidly adjusted to quickly pre‐screen already approved therapeutic antibodies or sera from vaccinated individuals for their ACE2 blocking activities against any emerging SARS‐CoV‐2 variants., A simple, inexpensive surrogate SARS‐CoV‐2 neutralization assay is introduced. Ectopic expression of the SARS‐CoV‐2 spike protein confers binding of Ramos‐Ig ‐ or HEK293 cells to plate‐bound recombinant human angiotensin‐converting‐enzyme‐2. This interaction can be blocked with reconvalescent or immune serum or with anti‐receptor‐binding domain antibodies. Bound spike‐expressing cells are quantitated with crystal violet.

Autoimmune lymphoproliferative syndrome (ALPS) is an uncommon disorder of Fas-mediated apoptosis that results in impaired lymphocyte death and, therefore, disturbed immune homeostasis. Besides presentation with lymphadenopathy and splenomegaly, patients with ALPS have a high incidence of autoimmune phenomena. To our knowledge, this is the first description of polyarteritis nodosa that includes numerous arterial aneurysms in a child with ALPS. Active vasculitis resolved after allogeneic hematopoietic stem cell transplantation. This report of polyarteritis nodosa associated with human ALPS supports previous findings in Fas-deficient mouse models that frequently develop vasculitic manifestations and suggests that apoptotic defects of lymphocytes may play a role in the pathophysiology of systemic vasculitis. Thus, patients with ALPS might be more susceptible to autoimmune vessel inflammation. This case furthermore emphasizes that even rare autoimmune manifestations should be considered and investigated in patients with immunodeficiencies, because that might help in planning treatment strategies for these patients.

Recent findings have demonstrated the properties of cell migration and cytokine secretion to be mutually exclusive and linked them to different functional subpopulations of dendritic cells (DCs). We studied human monocyte-derived DCs after stimulation with peptidoglycan (PGN), poly(I:C), lipopolysaccharide (LPS), and R848 (resiquimod) and found the resulting mature DCs to express CCR7, to migrate toward CCL19 and to be efficient primary interleukin (IL)-12 producers. Importantly, the potential for secondary production of large amounts of IL-12p70 in response to CD40 ligation was also preserved after stimulation by all Toll-like receptor (TLR) ligands. Differences between the TLR ligands were seen in the primary secretion of IL-12 and IL-23, in the survival of the DCs and in the expression of CD38. Finally, DCs stimulated by R848 were efficient in expanding peptide-specific CD8-positive T cells capable of peptide-specific target cell lysis. Together, our data suggest that TLR ligands induce the generation of mature DCs that integrate migratory and cytokine secretory capacity as well as cytotoxic T lymphocyte (CTL) stimulatory properties.

The culture of human monocyte-derived dendritic cells (DCs) is typically performed in media containing human or fetal calf serum, supplements with the potential to influence the cells phenotype and their functional properties. Published clinical trails based on serumfree cultured DCs reported the use of the commercially available medium AIMV. In this study, we directly compared DCs generated in AIMV medium ("AIMV/sf-DCs") with DCs generated in RPMI supplemented with 2% human serum ("RPMI/HS-DCs") in functional assays of potential relevance for vaccine application. Using TNF-alpha/PGE(2)/IL-1beta/IL-6 as maturation stimulus, AIMV/sf-DCs revealed to be comparable with RPMI/HS-DCs with regard to phenotypic expression of maturation markers, survival in vitro, migratory capacity and stimulation of lymphocyte proliferation except for CD1a which was expressed on a fraction of DCs only when cultured in serumfree AIMV medium. However, IL-12p70 production in response to Toll-like receptor (TLR) stimulating agents plus IFN-gamma was consistently lower in AIMV medium although also under serumfree culture conditions, nanogram quantities of IL-12 were produced. Together, DCs with functional characteristics important for in vivo application can be generated under defined serumfree conditions; however, medium and/or serum conditions appear to have strong influence on the production of relevant T cell differentiating cytokines.

Abstract 4696 Autoimmune lymphoproliferative syndrome (ALPS) is a rare immunologic disorder caused by defects in the Fas-induced programmed cell death pathway. Impaired lymphocyte apoptosis results in gradual lymphocyte accumulation and dysregulation of lymphocyte homeostasis. ALPS patients usually suffer from persistent generalized lymphadenopathy, hepato-splenomegaly, immune-mediated cytopenias, and other autoimmune phenomena. A 15-month-old boy was diagnosed with ALPS when he presented with typical symptoms. Lab exams showed a pancytopenia, elevated serum immunoglobulin levels, a peripheral expansion of double-negative T lymphocytes of up to 40% of TCRab+ T cells and impaired lymphocyte apoptosis. Molecular analysis confirmed the diagnosis of a type Ia ALPS by identifying a heterozygous Fas gene mutation (D260H). Despite treatment with repeated pulses of high-dose methylprednisolone, intravenous immunoglobulins, and mycophenolate mofetil (MMF) to control lymphoproliferation and recurrent pancytopenia his disease progressed. At three years of age he developed numerous arterial aneurysms of the iliac, mesenterial, renal, hepatic, right middle meningeal, brachial and femoral arteries up to 2.6 cm in diameter and lymphoproliferation resulting in paraplegia and right arm paresis. Stem cell transplantation was considered, and BMT from a 9/10 matched unrelated donor (MUD) after a reduced intensity conditioning regimen (CR) with Fludarabin (150 mg/m2), Melphalan (140 mg/m2/d) and ATG (60mg/kg) was performed using 5.22 × 106 CD34+ cells/kg body weight. However, graft failure had to be diagnosed on day +27. 53 days after the first BMT a PBSCT from the same donor after myeloablative CR using Busulfan (19.2 mg/kg/d i.v.), Etoposid (30 mg/kg/d), Cyclophosphamid (120 mg/kg/d) and ATG (60mg/kg) was performed (24.5 × 106 CD34+ cells/kg bw). Again, graft failure was seen. At day +55 a third HSCT using PBSCT from another 9/10 MUD after CR with Fludarabin i.v. (160 mg/m2), Thiotepa (5 mg/kg/d), 4 Gy total body irradiation (TBI) and campath (1 mg/kg) was performed. 10.8 × 106 CD34+ cells/kg bw were given intra osseous, 9.8 × 106 CD34+ cells/kg bw were given i.v. Engraftment was slow (Leukos 980 day +35), but chimerism showed 99 % donor cells. Two years later the patient is alive and well, with persistent engraftment and good hematological and immunological function. Arterial aneurysms stopped growing and some have thrombosed. This case illustrates some interesting points. Atypical and unusually severe manifestations of ALPS forced us to perform a HSCT in this patient. In severe ALPS stem cell engraftment is difficult to achieve as previously reported in the Fas deficient lpr mouse model. One reason might be the reduced ability of cytotoxic drugs to induce apoptosis in the Fas deficient recipient T cells. Thus, recipient T cells could persist and kill donor cells resulting in graft failure or rejection. Additionally, increased FasL expression on recipient cells could induce apoptosis in Fas bearing donor stem cells as shown in lpr mice. Also, trapping of infused stem cells in the extremely enlarged liver and spleen could have played a role. A 3rd attempt was therefore designed to overcome graft resistance and proved finally successful. The use of TBI together with campath possibly induced more T cell apoptosis than chemotherapy alone. The intra osseous application probably increased engraftment efficiency by avoiding trapping of stem cells in liver and spleen and possibly by induction of tolerance by intra osseous application as described before. Disclosures: No relevant conflicts of interest to declare.