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Unbiased dissection of T-cell receptor (TCR) repertoire diversity at the nucleotide level could provide important insights into human immunity. Here we show that TCR ligation-anchored-magnetically captured PCR (TCR-LA-MC PCR) identifies TCR α- and β-chain diversity without sequence-associated or quantitative restrictions in healthy and diseased conditions. TCR-LA-MC PCR identifies convergent recombination events, classifies different stages of cutaneous T-cell lymphoma in vivo and demonstrates TCR reactivation after in vitro cytomegalovirus stimulation. TCR-LA-MC PCR allows ultra-deep data access to both physiological TCR diversity and mechanisms influencing clonality in all clinical settings with restricted or distorted TCR repertoires. Immune system diversity is generated by V(D)J recombination, leading to clonal T-cell lineages. Here the authors investigate the events leading to T-cell diversity through the use of a modified PCR technique combined with deep sequencing.

Background: Primary cutaneous T-cell lymphomas (CTCLs) are rare lymphoproliferative malignancies characterized by significant morbidity and mortality in advanced disease stages. As curative approaches apart from allogeneic stem cell transplantation are lacking, establishing new treatment options, especially combination therapies, is crucial. Methods: This retrospective study included 11 patients with SS or MF receiving therapy with mogamulizumab in combination with ECP from four European expert centers. The response rates in the skin and blood as well as treatment use and adverse events (AE) were described. Results: 8/11 patients (73%) showed an overall response (OR) in the skin. The mean mSWAT decreased from 98.2 ± 40.8 to 34.6 ± 23.8. The overall response rate (ORR) in the blood was 64% with two complete responses. During combination therapy, the mean number of Sézary cells decreased from 3365.3 × 106/L before treatment to 1268.6 × 106/L. The mean minimum known period without progress was 7.2 months in the skin and 7.6 months in the blood. The most common AEs were mogamulizumab-associated rash (MAR) (45.5%), anemia (27.3%), lymphocytopenia (27.8%), and infusion related reaction (16.7%). No AE led to treatment discontinuation. Conclusions: Our study presents the combination of mogamulizumab and ECP as an effective therapy in the blood and skin in CTCL with good tolerability, similar to mogamulizumab monotherapy.

Primary cutaneous T cell lymphomas (CTCL) are a heterogeneous group of rare lymphoproliferative disorders originating in the skin. Extracorporeal photopheresis (ECP) is an established, effective and excellently tolerable CTCL therapy, that can also be applied for the treatment of graft vs. host disease (GvHD). However, the underlying molecular mechanisms of ECP have not yet been fully clarified and seem to be dependent on the underlying disease. In this study, peripheral blood samples collected from six CTCL and three GvHD patients were analyzed pre- and post-ECP within one treatment of ECP for short-term alterations in the cytokine and chemokine milieu in the plasma and the composition of the peripheral blood mononuclear cell (PBMC) subsets. In CTCL, the plasma profiling revealed a lower expression of IL-15, IL-17, ICOS and higher expression of IL-13 post-ECP compared to the pre-ECP samples. Additionally, ECP led to an increased expression of the cell death inducers Fas and TRAIL. Flow cytometry revealed a significant increase in the CD14+ monocytes post-ECP in the CTCL patients, and a tendency of higher CD3+CD4- cytotoxic T cells in GvHD patient. Therefore, one cycle of ECP can induce detectable alterations in the peripheral blood of both CTCL and GvHD patients. This study contributes to the elucidation of the molecular mechanisms of ECP therapy and the detection of potential biomarkers for therapeutic response to ECP.

Primary cutaneous T cell lymphomas (CTCL) are characterized by high relapse rates to initially highly effective therapies. Combination therapies have proven beneficial, particularly if they incorporate extracorporeal photopheresis (ECP). The NF-κB inhibitor dimethyl fumarate (DMF) has proven a new, effective drug in CTCL in a clinical phase II study. In vitro experiments with patient-derived SS cells and the CTCL cell lines HH, HuT 78, and SeAx revealed a synergistic effect of DMF and ECP on cell death induction in CTCL cells. Furthermore, an additional increase in the capacity to inhibit NF-κB in CTCL was detected for the combination treatment compared to DMF monotherapy. The same synergistic effects could be measured for ROS production via decreased Thioredoxin reductase activity and glutathione levels. Consequently, a cell death inhibitor screen indicated that the DMF/ECP combination treatment induces a variety of cell death mechanisms in CTCL. As a first step into clinical translation, 4 patients were already treated with the DMF/ECP combination therapy with an overall response rate of 100% and a time to next treatment in skin and blood of up to 57 months. Therefore, our study introduces the combination treatment of DMF and ECP as a highly effective and long-lasting CTCL therapy.

Wilson disease is caused by accumulation of Cu 2+ in cells, which results in liver cirrhosis and, occasionally, anemia. Here, we show that Cu 2+ triggers hepatocyte apoptosis through activation of acid sphingomyelinase (Asm) and release of ceramide. Genetic deficiency or pharmacological inhibition of Asm prevented Cu 2+ -induced hepatocyte apoptosis and protected rats, genetically prone to develop Wilson disease, from acute hepatocyte death, liver failure and early death. Cu 2+ induced the secretion of activated Asm from leukocytes, leading to ceramide release in and phosphatidylserine exposure on erythrocytes, events also prevented by inhibition of Asm. Phosphatidylserine exposure resulted in immediate clearance of affected erythrocytes from the blood in mice. Accordingly, individuals with Wilson disease showed elevated plasma levels of Asm, and displayed a constitutive increase of ceramide- and phosphatidylserine-positive erythrocytes. Our data suggest a previously unidentified mechanism for liver cirrhosis and anemia in Wilson disease.

To explore the functional significance of cGMP-dependent protein kinase type I (cGKI) in the regulation of erythrocyte survival, gene-targeted mice lacking cGKI were compared with their control littermates. By the age of 10 weeks, cGKI-deficient mice exhibited pronounced anemia and splenomegaly. Compared with control mice, the cGKI mutants had significantly lower red blood cell count, packed cell volume, and hemoglobin concentration. Anemia was associated with a higher reticulocyte number and an increase of plasma erythropoietin concentration. The spleens of cGKI mutant mice were massively enlarged and contained a higher fraction of Ter119⁺ erythroid cells, whereas the relative proportion of leukocyte subpopulations was not changed. The Ter119⁺ cGKI-deficient splenocytes showed a marked increase in annexin V binding, pointing to phosphatidylserine (PS) exposure at the outer membrane leaflet, a hallmark of suicidal erythrocyte death or eryptosis. Compared with control erythrocytes, cGKI-deficient erythrocytes exhibited in vitro a higher cytosolic Ca²⁺ concentration, a known trigger of eryptosis, and showed increased PS exposure, which was paralleled by a faster clearance in vivo. Together, these results identify a role of cGKI as mediator of erythrocyte survival and extend the emerging concept that cGMP/cGKI signaling has an antiapoptotic/prosurvival function in a number of cell types in vivo.

Background/Objectives: Advanced-stage mycosis fungoides (MF) and Sézary syndrome (SS) are aggressive forms of cutaneous T-cell lymphoma (CTCL) for which treatment options are limited and prognosis is poor. Brentuximab vedotin (BV), an anti-CD30 antibody–drug conjugate, has demonstrated high response rates in patients with CD30 expression ≥ 10%. However, data on its efficacy in cases with low CD30 expression (<10%) remain scarce. Methods: This retrospective analysis evaluated the real-world efficacy of BV in patients with advanced-stage MF/SS and low CD30 expression. A retrospective analysis was conducted on 32 patients across 11 German CTCL expert centers. All patients had advanced-stage MF or SS with CD30 expression < 10% and received BV at the standard dose. Treatment response was assessed using EORTC-ISCL criteria. Results: All patients had received prior systemic therapies (median: 3) with 36% having undergone prior mono- or polychemotherapy. The study population included 30 MF (stage IIB) and two SS cases. The overall response rate (ORR) in this population was 53.1% (17/32). A complete response (CR) was achieved in 12.5% (4/32), a partial response (PR) was achieved in 40.6% (13/32), stable disease (SD) was seen in 18.8% (6/32), and progressive disease (PD) was seen in 28.1% (9/32). The median progression-free survival (PFS) was 4.0 months (arithmetic mean: 6.38; range: 0.5–15.5), and the median time to next treatment (TTNT) was 7.25 months (arithmetic mean: 7.30; range: 2.00–15.5). Conclusions: BV demonstrated encouraging activity in heavily pretreated advanced MF/SS with low CD30 expression, achieving an ORR comparable to that observed in patients with higher CD30 levels. While response rates were similar, PFS was shorter. These findings suggest that BV remains a potential therapeutic option in this patient population and merits further prospective investigation.

Hemolytic uremic syndrome (HUS) is characterized by hemolytic anemia with fragmented erythrocytes, thrombocytopenia, and acute renal failure. Lack of complement inactivating factor H predisposes to the development of atypical HUS. Little is known about mechanisms linking complement activation with loss of erythrocyte integrity during HUS. Recent studies disclosed that increased cytosolic Ca2+ activity and cellular ceramide trigger programmed erythrocyte death or eryptosis, characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte surface. In the present study, we investigated whether eryptosis occurs during the course of HUS. To this end, erythrocytes from healthy volunteers were exposed to plasma from a patient with severe idiopathic recurrent HUS secondary to factor H depletion. Phosphatidylserine exposure (Annexin binding), cell volume (forward scatter), cytosolic Ca2+ activity (Fluo3 fluorescence), and ceramide formation [anti-ceramide antibody and enzymatic (diacylgycerol kinase) analysis] were determined. Exposure of erythrocytes to plasma from the patient, but not to plasma from healthy individuals, triggered Annexin binding. The effect of plasma on erythrocyte Annexin binding was abolished by plasmapheresis or filtration at 30 kDa. It was paralleled by formation of ceramide and increase of cytosolic Ca2+ activity. Enhanced Annexin binding of erythrocytes from healthy individuals was observed after exposure to plasma from three other patients with HUS. The proeryptotic effect of patient plasma was mimicked by exposure to the Ca2+ ionophore ionomycin, and eryptosis was potentiated in the presence of cell membrane-permeable C6-ceramide. Furthermore, in vitro complement activation similarly triggered erythrocyte phosphatidylserine exposure, an effect which was blunted by the addition of factor H. In conclusion, our present observations disclose a novel, pathophysiological, factor-H dependent mechanism leading to injury of erythrocytes during the course of hemolytic uremic syndrome.

Constitutively active NFκB promotes survival of many cancers, especially T-cell lymphomas and leukemias by upregulating antiapoptotic proteins such as inhibitors of apoptosis (IAPs) and FLICE-like inhibitory proteins (cFLIPs). IAPs and cFLIPs negatively regulate the ripoptosome, which mediates cell death in an apoptotic or necroptotic manner. Here, we demonstrate for the first time, that DMF antagonizes NFκB by suppressing Thioredoxin-1 (Trx1), a major regulator of NFκB transcriptional activity. DMF-mediated inhibition of NFκB causes ripoptosome formation via downregulation of IAPs and cFLIPs. In addition, DMF promotes mitochondrial Smac release and subsequent degradation of IAPs, further enhancing cell death in tumor cells displaying constitutive NFκB activity. Significantly, CTCL patients treated with DMF display substantial ripoptosome formation and caspase-3 cleavage in T-cells. DMF induces cell death predominantly in malignant or activated T-cells. Further, we show that malignant T-cells can die by both apoptosis and necroptosis, in contrast to resting T-cells, which are restricted to apoptosis upon DMF administration. In summary, our data provide new mechanistic insight in the regulation of cell death by targeting NFκB via Trx1 in cancer. Thus, interference with Trx1 activity is a novel approach for treatment of NFκB-dependent tumors.

Microangiopathy with subsequent organ damage represents a major complication in several diseases. The mechanisms leading to microvascular occlusion include von Willebrand factor (VWF), notably the formation of ultra-large von Willebrand factor fibers (ULVWFs) and platelet aggregation. To date, the contribution of erythrocytes to vascular occlusion is incompletely clarified. We investigated the platelet-independent interaction between stressed erythrocytes and ULVWFs and its consequences for microcirculation and organ function under dynamic conditions. In response to shear stress, erythrocytes interacted strongly with VWF to initiate the formation of ULVWF/erythrocyte aggregates via the binding of Annexin V to the VWF A1 domain. VWF-erythrocyte adhesion was attenuated by heparin and the VWF-specific protease ADAMTS13. In an in vivo model of renal ischemia/reperfusion injury, erythrocytes adhered to capillaries of wild-type but not VWF-deficient mice and later resulted in less renal damage. In vivo imaging in mice confirmed the adhesion of stressed erythrocytes to the vessel wall. Moreover, enhanced eryptosis rates and increased VWF binding were detected in blood samples from patients with chronic renal failure. Our study demonstrates that stressed erythrocytes have a pronounced binding affinity to ULVWFs. The discovered mechanisms suggest that erythrocytes are essential for the pathogenesis of microangiopathies and renal damage by actively binding to ULVWFs.