Background: Protein S (PS) is an important, nonenzymatic cofactor with clinical relevance in coagulation disorders. Hereditary deficiency of PS is caused by pathogenic variants in the PROS1 gene, which encodes this protein. Identifying the presence and assessing the pathogenicity of PROS1 variants is essential for the effective management of patients with hereditary PS deficiency. Objectives: The aim of this study was to identify PROS1 variants in patients with suspected hereditary PS deficiency and to evaluate their pathogenicity. Methods: The PROS1 coding sequence, including adjacent intron-exon boundaries, was analyzed in 276 patients with suspected hereditary PS deficiency using next-generation sequencing. Identified variants were evaluated based on existing literature, when available, or using bioinformatic prediction tools. The predicted pathogenicity was then compared with the patients' PS activity levels. Results: Forty-eight distinct variants were identified in 101 patients. Twenty-seven of these variants have been previously described in the literature, and their pathogenicity was categorized based on all available evidence. Of the remaining 21 variants, 11 are listed in ClinVar and/or dbSNP; 10 are novel. The potential pathogenicity of these variants, as well as possible explanations for PS deficiency in patients without an identified variant, are discussed. Conclusion: In silico prediction tools are useful for a first assessment of pathogenicity for novel PROS1 variants. For a final evaluation of whether a variant is causative for hereditary PS deficiency, a comprehensive characterization is indispensable.

The identification of novel compounds capable of stimulating or suppressing immune responses is critical for advancing immunotherapy and improving our understanding of immune regulation. However, traditional screening approaches are often limited by low throughput, poor physiological relevance, and insufficient resolution to detect subtle immunomodulatory effects. In this study, we introduce a validated and robust multiplexed flow cytometry-based functional screening assay that enables efficient and precise identification of immunomodulatory compounds, while addressing the key limitations of conventional methods. By using human peripheral blood mononuclear cells (PBMCs) and defined stimulation conditions, the assay mimics complex immune interactions in a physiologically relevant context. It simultaneously monitors T- and B-cell responses across multiple functional readouts, including T-cell proliferation (carboxyfluorescein succinimidyl ester [CFSE]), activation (CD25, CD71), and B-cell costimulatory marker expression (CD86), enabling an in-depth assessment of immune modulation within heterogenous cell populations. The incorporation of robust statistical validation using the Z-factor further supports assay reliability and reproducibility. In a pilot screen with 295 immunology-related compounds, the assay demonstrated its ability to identify known immunomodulators with distinct functional profiles. This multiplex functional screening approach bridges the gap between mechanistic immunology and drug discovery by enabling systematic identification and characterization of immunotherapeutic candidates, while providing new insights into the complex dynamics of immune regulation.

Bispecific antibodies are used for the treatment of hematological malignancies as well as solid tumors. One of their main effector mechanisms is the recruitment of effector cells such as CD8+ T cells and CD16A+ NK cells to tumor cells. Bispecific innate cell engagers (ICE®) harnessing CD16A+ NK cells have been shown to induce significant tumor cell lysis in preclinical models, translating to promising signs of clinical activity together with a well-managed safety profile. However, how killing of tumor cells by NK cells influences other innate immune cells in the tumor microenvironment, such as dendritic cells (DCs), instrumental in bridging innate and adaptive tumor immunity, is largely unknown. Thus, we here analyzed whether antibody-dependent cell-mediated cytotoxicity by NK cells affected human DC subpopulations. We could show that killing of tumor cells leads to a strong activation of human conventional DCs type 1 (cDC1), DC2, and DC3 with enhanced expression of co-stimulatory molecules as well as the secretion of proinflammatory cytokines. Further, DC subpopulations as well as surviving tumor cells showed increased expression of the immunoregulatory molecule PD-L1 that is known to dampen T-cell immunity. Nevertheless, ADCC boosted the capacity of cDC1 and DC2 to prime naïve T cell responses but not of DC3. Thus, our data suggests that the therapy with bispecific antibodies targeting NK cells may have the potential to facilitate adaptive antitumor immune responses via activation of cDC1 and DC2.

Background:Extracorporeal photopheresis (ECP) is a therapy indicated for various T cell-mediated conditions, including cutaneous T-cell lymphoma (CTCL), graft-versus-host disease (GVHD), and solid organ transplant rejection. ECP comprises the treatment of patients' leukocytes with 8-methoxypsoralen and ultraviolet-A light followed by autologous reinfusion. ECP exerts therapeutic immune-stimulatory effects in CTCL and immune regulatory effects in GVHD and solid organ transplant rejection. Besides cellular mediators, secreted molecules can contribute to ECP's therapeutic effect. Methods:We conducted a comprehensive review of the literature on ECP-induced secreted factors and their immunomodulatory roles. Results:8-Methoxypsoralen/ultraviolet-A treatment drives leukocyte apoptosis, resulting in the release of damage-associated molecular patterns that promote apoptotic cell phagocytosis by dendritic cells (DCs) and promote or impair DC maturation. In CTCL, the increased production of proinflammatory cytokines in photopheresates, including interferon-γ, interleukin (IL)-2, tumor necrosis factor-α, IL-1β, and IL-8, is linked to antitumor responses. Conversely, ECP upregulates anti-inflammatory cytokine production in photopheresates from GVHD patients' cells. Upon reinfusion of photopheresates containing anti-inflammatory factors, untreated immature DCs are converted to tolerogenic DCs with increased IL-10 and transforming growth factor-β secretion and regulatory T cell-inducing functions. In allograft models, ECP increases IL-4, IL-10, and IL-13, which reduce allograft rejection. Moreover, ECP influences the level of immunomodulatory metabolites and the composition of exosomes. However, further research, for example, using multi-omics approaches, are needed to provide a more comprehensive picture of the ECP-induced secretome and to identify relevant factors that could contribute to ECP's therapeutic effects. Conclusions:ECP induces the release of different pro/anti-inflammatory factors in different preexisting conditions that determine different DC maturation status and immunomodulatory effects.

IntroductionExtracorporeal photopheresis (ECP) is an immunomodulatory treatment option for different T cell-mediated diseases such as cutaneous T cell lymphoma (CTCL) and chronic graft-versus-host disease (GvHD). While in CTCL the polarization of T cells is shifted towards T helper cells type 1 (TH1) and an immune response against the lymphoma is induced, ECP in GvHD rather leads to the expansion of regulatory T cells (Treg). How ECP regulates the immune response dependent on the underlying disease is still not exactly known. As dendritic cells (DCs) are crucial regulators of the immune response, it is supposed that they are key players in the immunomodulatory effects of ECP. However, due to the scarcity of primary DCs in blood, research has focused on in vitro-generated monocyte-derived DCs so far.MethodsHere, we present for the first time how the primary human blood DC subpopulations, i.e., conventional DCs type 1 (cDC1), cDC2, DC3, and plasmacytoid DCs (pDC), directly isolated from blood of healthy donors, respond to in vitro ECP treatment. ResultsWe demonstrate that the exposure to 8-methoxypsoralen and UV-A light irradiation induces apoptosis in Toll-like receptor ligand-activated cDC1 and pDC as well as - to a minor extent - in steady state cDC1, cDC2, and DC3. However, the selective effect of ECP on viability of DC subpopulations was dependent on culture duration (18h vs. 42h) as well as condition (steady state vs. TLR ligand activated). Further, ECP modulates the expression of the co-stimulatory and co-regulatory molecules CD40, CD86, and PD-L1 on DC subpopulations. While ECP did not affect the T cell stimulatory capacity of cDC2 and DC3, ECP-treated cDC1 and - to a minor extent - pDC showed reduced activation of memory T cells and diminished secretion of TH1- and TH17-associated cytokines. ConclusionThus, especially blood cDC1 are direct targets of ECP and the reduction of their T cell stimulatory capacity might contribute to the clinical efficacy observed in chronic GvHD patients.

OBJECTIVE:Pathogenic variants in COL4A1 and COL4A2 genes encoding α-chains of type IV collagen in basement membranes of vessels and soft organs are associated with multi-system phenotypes affecting brain, eye, kidney and muscle. To date, several pathogenic germline variants, either de novo or inherited, have been reported, with phenotype descriptions covering a few hundred individuals. Characteristic brain manifestations include cerebral small vessel disease (cSVD), causing microangiopathy with leukoaraiosis, ischemic stroke events, and intracerebral hemorrhages in infancy to adulthood. Here, we report a case with a novel COL4A1 variant, presenting with cSVD at juvenile age. MATERIALS AND METHODS:A 21-year-old female developed two stroke episodes involving hemiparesis and ocular palsy. Head imaging revealed recurrent supra- and infratentorial ischemic and hemorrhagic brain injuries, subcortical microbleeds and white matter lesions. Porencephalic ventricle dysmorphology was also obvious. Expanded stroke diagnostics included molecular trio-exome sequencing, analyses of mitochondrial DNA and of copy number variants. RESULTS:Diagnostic measures for vascular, cardiac and coagulation disorders did not show pathologies. Genetic analyses revealed a novel, likely pathogenic missense variant in the triple-helical region of COL4A1 not detectable in the patient's parents. CONCLUSIONS:We inferred cSVD of monogenic origin, based on a so far undescribed COL4A1 de novo variant. Increased awareness and reporting of COL4A variants can elucidate cryptogenic juvenile stroke and improve patient management in the context of precision medicine.

Introduction: Immune checkpoint inhibitors (ICIs) have revolutionized classical treatment approaches of various cancer entities, but are also associated with a number of side effects. One of these may be life-threatening clotting disorders with the risk of thrombotic or hemorrhagic complications, the mechanisms of which are still poorly understood. In the present study, we analyzed the direct effects of pembrolizumab, nivolumab, and ipilimumab on platelet aggregation as well as plasma coagulation followed by fibrinolysis in an ex vivo model. Methods: Microplate spectrometry was used to analyze aggregation, coagulation, and fibrinolysis in platelet-free (PFP) and platelet-rich (PRP) healthy donor plasma samples treated with pembrolizumab, nivolumab, ipilimumab, and appropriate isotype controls. Aggregation was induced by TRAP-6. Clotting of PFP and PRP followed by lysis was initiated with a tissue factor in a mixture of phosphatidylserine:phosphatidylcholine and the addition of t-PA. Among other parameters, the area under the curve (AUC) was used to compare the effect of ICIs on aggregation, coagulation, and fibrinolysis. Results: Upon direct contact with platelets, pembrolizumab stimulated platelet aggregation in PRP, while nivolumab and ipilimumab promoted disaggregation with corresponding changes in the AUC. Pembrolizumab and nivolumab, both PD-1 receptor inhibitors, had no effect on the plasma coagulation cascade. Ipilimumab, a CTLA-4 receptor inhibitor, significantly increased the rate of PRP clotting. When clotting was followed by lysis, all ICIs were found to prolong the growth of the PRP-derived fibrin clot and delay its elimination. This was manifested by an increase in AUC relative to control PRP. Conclusion: This study characterizes the potential impact of pembrolizumab, nivolumab, and ipilimumab on hemostasis. Nivolumab and ipilimumab are able to reduce aggregation and increase the procoagulant properties of platelets, which can cause side effects associated with hemostatic imbalance leading to thrombosis or bleeding. The observed ICI-specific effects may contribute to our understanding of the mechanisms by which ICI affects platelets and suggest how, in a clinical setting, to reduce coagulation disorders during ICI treatment in the future.

BACKGROUND:The recently identified PROS1 mutation Protein S Erlangen c.1904T>C, resulting in amino acid exchange F635S, is associated with severe quantitative protein S (PS) deficiency and clinical thrombosis. It was hypothesized that this deficiency is due to a secretion defect [1]. This report aims to further elucidate the potential secretion defect of PS Erlangen.METHODS:Coding sequences (CDS) of wild type (WT) PROS1 (encoding PS) and mutated PROS1c.1904T>C (encoding PSF635S) were cloned in front of the CDS of green fluorescent protein (GFP), and the respective plasmids were introduced into HEK293T cells. PROS1-GFP and PROS1c.1904T>C-GFP expressing HEK293T cell lines were analyzed by confocal laser scanning microscopy and western blot for cellular proteins and proteins secreted to the growth medium.RESULTS:Western blot analysis revealed a significantly reduced secretion of PSF635S compared to WT PS. This observation was confirmed by the detection of mutant PSF635S-GFP fusion exclusively in the endoplasmic reticulum (ER), while PS-GFP passed through the entire secretory pathway, as indicated by the localization within both the ER and Golgi apparatus.CONCLUSIONS:The Protein S Erlangen mutation results in type I PS deficiency caused by a secretion defect.

ZusammenfassungDie extrakorporale Photopherese (ECP) ist eine Behandlungsmethode, die bei Patienten mit T-Zell-vermittelten Erkrankungen wie der Graft-versus-Host-Disease (GVHD) seit Jahren erfolgreich klinisch angewandt wird. Unter den Patienten nach soliden Organtransplantationen zeigen lungentransplantierte Patienten bei supportivem Einsatz der ECP-Therapie auch ein gutes klinisches Ansprechen bei Vorliegen einer Bronchiolitis obliterans als Hinweis auf eine chronische Abstoßungsreaktion (Nachweis eines stabilen forcierten exspiratorischen Einsekundenvolumens [FEV1]). Studien zeigen, dass sich durch den therapeutischen Einsatz der ECP eine Verlangsamung der chronischen Abstoßung nach Lungentransplantation (LTX) erreichen lässt.Bei einer 39-jährigen Patientin mit Bronchiolitis-obliterans-Syndrom (BOS) (Stadium 2) als Zeichen einer chronischen Funktionsstörung des Lungen-Allotransplantats nach sequenzieller Doppellungentransplantation wurde eine ECP-Behandlung empfohlen. Die ECP wurde ambulant mit einer Frequenz von 2 ECP-Behandlungen an 2 aufeinanderfolgenden Tagen alle 4 Wochen durchgeführt. Die Behandlung wurde offline durchgeführt und die MCS+(Haemonetics) für die Leukapherese verwendet. Das behandelte Volumen lag zwischen 1,0 und 1,5 Gesamtkörpervolumen (TBV). Als Strahlungsquelle diente ein UVA-PIT-Bestrahlungsgerät. Dem vor der Bestrahlung gewonnenen Leukapheresat wurde Methoxsalen in einer Dosierung von 300 ng/ml zugesetzt. Das Leukapheresat mit einem Hämatokrit < 3% wurde mit UVA-Licht (Bestrahlungsdosis > 2 J/cm2)bestrahlt und nach der Behandlung retransfundiert. Es wurde eine Verlangsamung der chronischen Abstoßungsreaktion bei Bronchiolitis obliterans und eine Stabilisierung der Lungenfunktion erreicht, die sich in einem stabilen FEV1 zeigte.

The null allele HLA-C*04:09N differs from HLA-C*04:01 in a frameshift mutation within its cytoplasmic domain, resulting in translation of 32 additional amino acids that are assumed to prevent cell surface expression. However, we recently identified a multiple myeloma-reactive T-cell receptor (TCR) that appeared to recognize antigen presented on HLA-C*04:09N and encouraged us to ask whether HLA-C*04:09N, albeit not easily detectable at the cell surface, can present antigen sufficient for T-cell activation. We generated two HLA-class I-deficient cell lines, re-expressed HLAC* 04:09N, detected HLA expression by flow cytometry, and tested for T-cell activation using a cytomegalovirus peptide- specific HLA-C*04:01-restricted TCR. In both cell lines, HLA-C*04:09N expression was detectable at the cell surface and could be enhanced by IFN-γ exposure. Recombinant HLA-C*04:09N expression was sufficient for T-cell activation in vitro, which could be blocked by an HLA-class I-specific antibody, suggesting HLA-TCR interaction at the cell surface. Peripheral blood mononuclear cells isolated from an individual who physiologically expressed HLA-C*04:09N triggered peptide-specific T-cell activation, confirming our results with cells with natural HLA expression levels. In conclusion, we present peptide-specific HLA-C*04:09N-restricted T-cell activation and suggest consideration of this allele in the appropriate clinical context, such as allogeneic stem cell transplantation, or in the setting of cellular therapy.

Human umbilical cord blood (UCB) represents a unique resource for hematopoietic stem cell transplantation for children and patients lacking suitable donors. UCB harbors a diverse set of leukocytes such as professional APCs, including monocytes, that could act as a novel source for cellular therapies. However, the immunological properties of UCB monocytes and monocyte-derived dendritic cells (MoDCs) are not fully characterized. In this study, we characterized the phenotype and functions of UCB-MoDCs to gauge their potential for future applications. UCB exhibited higher frequencies of platelets and lymphocytes as well as lower frequencies of neutrophils in comparison with adult whole blood. Leukocyte subset evaluation revealed significantly lower frequencies of granulocytes, NK cells, and CD14+CD16− monocytes. Surface marker evaluation revealed significantly lower rates of costimulatory molecules CD80 and CD83 while chemokine receptors CCR7 and CXCR4, as well as markers for Ag presentation, were similarly expressed. UCB-MoDCs were sensitive to TLR1–9 stimulation and presented quantitative differences in the release of proinflammatory cytokines. UCB-MoDCs presented functional CCR7-, CXCR4-, and CCR5-associated migratory behavior as well as adequate receptor- and micropinocytosis-mediated Ag uptake. When cocultured with allogeneic T lymphocytes, UCB-MoDCs induced weak CD4+ T lymphocyte proliferation, CD71 expression, and release of IFN-γ and IL-2. Taken together, UCB-MoDCs present potentially advantageous properties for future medical applications.

Extracorporeal photopheresis (ECP) is a treatment method that has successfully been used clinically for years in patients with T-cell mediated disease, such as the graft-versus-host disease (GVHD). Among patients after solid organ transplantation, lung transplant patients also show a good clinical response with supportive ECP therapy in the presence of bronchiolitis obliterans as an indication of a chronic rejection reaction (evidence of a stable forced expiratory one-second volume [FEV1]). Studies have confirmed a deceleration of the chronic rejection reaction in patients with lung transplantation when using extracorporeal photopheresis therapeutically. In a 39-year-old female patient with bronchiolitis obliterans syndrome (BOS) (stage 2) as a sign of chronic lung allograft dysfunction after sequential double lung transplantation, a continued ECP treatment was suggested. The ECP has been carried out on an outpatient basis at a frequency of 2 ECP treatments on 2 consecutive days every 4 weeks. The treatment was performed offline and MCS+(Haemonetics) was used for leukapheresis. The processed volume was between 1.0 and 1.5 total body volume (TBV). The radiation source used was an UVA-PIT irradiation device. Methoxsalen at a dosage of 300 ng/ml was added to the leukapheresate obtained prior to irradiation. The leukapheresate with a hematocrit < 3% was irradiated with UVA light (irradiation dose > 2 J/cm(2)) and retransfused after treatment. A slowing down of the chronic rejection reaction in bronchiolitis obliterans was achieved and a stabilization of the lung function, as indicated by a stable FEV1.

Acquired aplastic anemia is a bone marrow failure syndrome characterized by hypocellular bone marrow and peripheral blood pancytopenia. Frequent clinical responses to calcineurin inhibition and antithymocyte globulin strongly suggest critical roles for hematopoietic stem/progenitor cell-reactive T-cell clones in disease pathophysiology; however, their exact contribution and antigen speci fi cities remain unclear. We determined differentiation states and targets of dominant T-cell clones along with their potential to eliminate hematopoietic progenitor cells in the bone marrow of 15 patients with acquired aplastic anemia. Single-cell sequencing and immunophenotyping revealed oligoclonal expansion and effector differentiation of CD8 + T-cell compartments. We reexpressed 28 dominant T-cell receptors (TCRs) of 9 patients in reporter cell lines to determine reactivity with (1) in vitro-expanded CD34 + bone marrow, (2) CD34 - bone marrow, or (3) peptide pools covering immunodominant epitopes of highly prevalent viruses. Besides 5 cytomegalovirus-reactive TCRs, we identi fi ed 3 TCRs that recognized antigen presented on hematopoietic progenitor cells. T cells transduced with these TCRs eliminated hematopoietic progenitor cells of the respective patients in vitro. One progenitor cell-reactive TCR (11A5) also recognized an epitope of the EpsteinBarr virus-derived latent membrane protein 1 (LMP1) presented on HLA-A*02:01. We identi fi ed 2 LMP1-related mimotopes within the human proteome as activating targets of TCR 11A5, providing proof of concept that molecular mimicry of viral and self-epitopes can drive T cell-mediated elimination of hematopoietic progenitor cells in aplastic anemia.

Background Antithrombin (AT) is an important anticoagulant in hemostasis. We describe here the characterization of a novel AT mutation associated with clinically relevant thrombosis. A pair of sisters with confirmed type I AT protein deficiency was genetically analyzed on suspicion of an inherited SERPINC1 mutation. A frameshift mutation, c.1247dupC, was identified and the effect of this mutation was examined on the cellular and molecular level. Methods Plasmids for the expression of wild-type (WT) and mutated SERPINC1 coding sequence (CDS) fused to green fluorescent protein ( GFP ) or hemagglutinin ( HA ) tag were transfected into HEK293T cells. Subcellular localization and secretion of the respective fusion proteins were analyzed by confocal laser scanning microscopy and Western blot. Results The c.1247dupC mutation results in a frameshift in the CDS of the SERPINC1 gene and a subsequently altered amino acid sequence (p.Ser417LysfsTer48). This alteration affects the C-terminus of the AT antigen and results in impaired secretion as confirmed by GFP- and HA-tagged mutant AT analyzed in HEK293T cells. Conclusion The p.Ser417LysfsTer48 mutation leads to impaired secretion, thus resulting in a quantitative AT deficiency. This is in line with the type I AT deficiency observed in the patients.

Immune checkpoint inhibitor (ICI) therapies effectively treat a broadening spectrum of cancer entities but induce various immune-related side effects (irAEs). Recent reports suggest a correlation between ICI-induced systemic inflammation and thromboembolic events as well as an increased effectiveness by coadministration of anticoagulants. With cancer patients having a higher risk of thrombotic events per se, it is crucial to dissect and characterize the mechanisms that cause pro-coagulative effects induced by systemic tumor therapies and their potential interplay with anti-tumor response. A total of 31 patients with advanced skin cancer treated with either ICIs (n = 24) or BRAF/MEK inhibitors (n = 7) were longitudinally assessed for blood and coagulation parameters before as well as 7, 20 and 40 days after initiation of systemic tumor therapy. Changes were analyzed and compared between both groups. In addition, the influence of coagulation parameters on progression-free, recurrence-free and overall survival was investigated. The ICI cohort presented significantly increased factor VIII activity after one week of therapy (p 0.0225); while, protein S activity was reduced during the whole observation period. Additionally, von Willebrand factor activity and tissue factor concentrations increased under immunotherapy. Similar changes occurred under BRAF/MEK inhibitor therapy (BRAF/MEKi). Increased baseline levels of von Willebrand factor antigen and factor VIII:C before the start of ICI therapy correlated with a significantly higher risk of recurrence for patients receiving adjuvant immunotherapy. The findings suggest the induction of a pro-coagulant state under ICI and BRAF/MEKi and a role of coagulation parameters in the efficacy of ICI therapies.

Introduction:Parvovirus B19 transmitted by umbilical cord blood (UCB) products may cause severe disease in allogenic hematopoietic stem cell transplant recipients. Thus, commercially available nucleic acid test (NAT) assays for highly sensitive detection of parvovirus B19 DNA validated for the specimen cord blood plasma (CBP) are required to avoid parvovirus B19 transmission by umbilical hematopoietic stem cell preparations. Methods:The multiplex cobas DPX NAT assay was validated for detection of parvovirus B19 DNA in CBP derived from citrate anticoagulated UCB units which have been processed by the Rubinstein method. In total, 363 retained CBP samples pretested negative for parvovirus B19 DNA were prepared for analyzing sensitivity, specificity, and interference of that NAT assay. The 3rd WHO International Standard for parvovirus B19 DNA was used for determining the 95% limit of detection (LOD95) by probit analysis. Results:The validation of the parvovirus B19 NAT assay for CBP demonstrated high sensitivity, specificity, intra- and inter-assay precision. Dilution series and replicate analyses showed a high linearity of the assay with a coefficient of determination above 0.99 and revealed a LOD95 of 17 International Units (IU)/mL (95% confidence interval, 14-44 IU/mL) for parvovirus B19 DNA in CBP samples. Conclusion:The validation of a commercially available parvovirus B19 NAT assay for the specimen CBP demonstrated a high assay performance fulfilling German guidelines and international regulations.

OBJECTIVE:Ulcerative colitis (UC) is a chronic, debilitating immune-mediated disease driven by disturbed mucosal homeostasis, with an excess of intestinal effector T cells and an insufficient expansion of mucosal regulatory T cells (Tregs). We here report on the successful adoptive transfer of autologous, ex vivo expanded Tregs in a patient with refractory UC and associated primary sclerosing cholangitis (PSC), for which effective therapy is currently not available.DESIGN:The patient received a single infusion of 1×106 autologous, ex vivo expanded, polyclonal Tregs per kilogram of body weight, and the clinical, biochemical, endoscopic and histological responses were assessed 4 and 12 weeks after adoptive Treg transfer.RESULTS:The patient showed clinical, biochemical, endoscopic and histological signs of response until week 12 after adoptive Treg transfer, which was associated with an enrichment of intestinal CD3+/FoxP3+ and CD3+/IL-10+ T cells and increased mucosal transforming growth factor beta and amphiregulin levels. Moreover, there was marked improvement of PSC with reduction of liver enzymes. This pronounced effect lasted for 4 weeks before values started to increase again.CONCLUSION:These findings suggest that adoptive Treg therapy might be effective in refractory UC and might open new avenues for clinical trials in PSC.TRIAL REGISTRATION NUMBER:NCT04691232.

The "biological identity" of nanoparticles (NPs) is governed by a shell consisting of various biomolecules that is formed upon exposure to biological media, the so-called biomolecule corona. Consequently, supplementation of cell culture media with e.g. different sera is likely to affect interactions between cells and NPs ex-vivo, especially endocytosis. We aimed to investigate the differential impact of human and fetal-bovine serum on the endocytosis of poly (lactic-co-glycolic acid) NPs by human peripheral blood mononuclear cells via flow cytometry. Furthermore, we employed different methods to inhibit endocytosis, providing mechanistic insights. The resulting biomolecule corona was characterized via denaturing gel electrophoresis. We found profound differences between human and fetal bovine serum regarding the endocytosis of fluorescently labeled PLGA nanoparticles by different classes of human leukocytes. Uptake by B-lymphocytes was particularly sensitive. We further present evidence, that these effects are mediated by a biomolecule corona. We demonstrate to our knowledge for the first time that the complement is an important contributor to the endocytosis of non-surface-engineered PLGA-nanoparticles prepared via emulsion solvent evaporation by human immune cells. Our data demonstrates that results obtained with xenogeneic culture supplements such as fetal bovine serum may have to be interpreted with caution.