Profile of the single-use, multiple-pass protein A adsorber column in immunoadsorption.

BACKGROUND AND OBJECTIVES: Immunoadsorptions (IA) are used to remove autoantibodies from the plasma in autoimmune disorders. In this study, we evaluated the effects of a single-use, recombinant staphylococcal protein A-based immunoadsorber on blood composition of the patient. MATERIALS AND METHODS: In a cohort of patients with myasthenia gravis or stiff-person syndrome, essential parameters of blood cell count, coagulation, clinical chemistry or plasma proteins and immunoglobulins (Ig) were measured before and after IA (n = 11). RESULTS: In average, IA reduced the levels of total IgG, IgG1, IgG2 and IgG4 by approximately 60%, the acetylcholine receptor autoantibody levels by more than 70%. IgG3, IgA or IgM were diminished to a lower extent. In contrast to fibrinogen or other coagulation factors, the column markedly removed vitamin K-dependent coagulation factors II, VII, IX and X by approximately 40%-70%. Accordingly, international normalized ratio and activated partial thromboplastin time were increased after IA by 59.1% and 32.7%, respectively. Coagulation tests almost returned to baseline values within 24 h. Blood cell count, electrolytes, total protein or albumin were not essentially affected. No clinical events occurred. CONCLUSION: The single-use, multiple-pass protein A adsorber column is highly efficient to remove IgG1, IgG2 and IgG4 or specific acetylcholine receptor autoantibodies from the plasma. Coagulation parameters should be monitored, since the column has the capacity to largely reduce vitamin K-dependent factors.

Natural and cryptic peptides dominate the immunopeptidome of atypical teratoid rhabdoid tumors.

BACKGROUND: Atypical teratoid/rhabdoid tumors (AT/RT) are highly aggressive CNS tumors of infancy and early childhood. Hallmark is the surprisingly simple genome with inactivating mutations or deletions in the SMARCB1 gene as the oncogenic driver. Nevertheless, AT/RTs are infiltrated by immune cells and even clonally expanded T cells. However, it is unclear which epitopes T cells might recognize on AT/RT cells. METHODS: Here, we report a comprehensive mass spectrometry (MS)-based analysis of naturally presented human leukocyte antigen (HLA) class I and class II ligands on 23 AT/RTs. MS data were validated by matching with a human proteome dataset and exclusion of peptides that are part of the human benignome. Cryptic peptide ligands were identified using Peptide-PRISM. RESULTS: Comparative HLA ligandome analysis of the HLA ligandome revealed 55 class I and 139 class II tumor-exclusive peptides. No peptide originated from the SMARCB1 region. In addition, 61 HLA class I tumor-exclusive peptide sequences derived from non-canonically translated proteins. Combination of peptides from natural and cryptic class I and class II origin gave optimal representation of tumor cell compartments. Substantial overlap existed with the cryptic immunopeptidome of glioblastomas, but no concordance was found with extracranial tumors. More than 80% of AT/RT exclusive peptides were able to successfully prime CD8+ T cells, whereas naturally occurring memory responses in AT/RT patients could only be detected for class II epitopes. Interestingly, >50% of AT/RT exclusive class II ligands were also recognized by T cells from glioblastoma patients but not from healthy donors. CONCLUSIONS: These findings highlight that AT/RTs, potentially paradigmatic for other pediatric tumors with a low mutational load, present a variety of highly immunogenic HLA class I and class II peptides from canonical as well as non-canonical protein sources. Inclusion of such cryptic peptides into therapeutic vaccines would enable an optimized mapping of the tumor cell surface, thereby reducing the likelihood of immune evasion.

Peptide receptor radionuclide therapy as a new tool in treatment-refractory sarcoidosis - initial experience in two patients.

Sarcoidosis is a multisystem granulomatous disorder of unknown etiology that can involve virtually all organ systems. Whereas most patients present without symptoms, progressive and disabling organ failure can occur in up to 10% of subjects. Somatostatin receptor (SSTR)-directed peptide receptor radionuclide therapy (PRRT) has recently received market authorization for treatment of SSTR-positive neuroendocrine tumors. Methods: We describe the first case series comprising two patients with refractory multi-organ involvement of sarcoidosis who received 4 cycles of PRRT. Results: PRRT was well-tolerated without any acute adverse effects. No relevant toxicities could be recorded during follow-up. Therapy resulted in partial response accompanied by a pronounced reduction in pain (patient #1) and stable disease regarding morphology as well as disease activity (patient #2), respectively. Conclusion: Peptide receptor radionuclide therapy in sarcoidosis is feasible and might be a new valuable tool in patients with otherwise treatment-refractory disease. Given the long experience with and good tolerability of PRRT, further evaluation of this new treatment option for otherwise treatment-refractory sarcoidosis in larger patient cohorts is warranted.

Boost and loss of immune responses against tumor-associated antigens in the course of pregnancy as a model for allogeneic immunotherapy.

Donor-derived immunity against tumor-associated antigens (TAAs) may exert selective antileukemic activity reprieving the allogeneic recipient from graft-versus-host disease. As TAAs are highly expressed in placental tissues we hypothesized that pregnancy could drive respective immunity in healthy individuals. Thus, we investigated the frequency and level of immune responses against clinically relevant TAAs in 114 blood donors and 44 women during their first pregnancy. Quantitative reverse-transcription polymerase chain reaction was employed to detect low levels of interferon-γ after primary peptide stimulation of CD8(+) T lymphocytes. In blood donors, primary immune responses of low and/or high avidity were found against WT1 (15%), MUC1 (14%), PRAME (7%), and HER2/neu (5%) and exerted killing functions against leukemic cells. Men had higher responses than women, likely due to gonadal cancer-testis-antigen expression. Interestingly, a history of prior delivery was not associated with increased responses, whereas the strongest responses during pregnancy were found in early trimesters to disappear after delivery. This boost and loss of TAA-specific immunity suggests that virtually every donor harbors the potential to mount antileukemic immune responses in a recipient. However, in the absence of the driving target and a permissive environment, they are short-lived and thus require supplemental strategies such as vaccination or immunomodulation to facilitate their persistence.

A comprehensive analysis of primary acute myeloid leukemia identifies biomarkers predicting susceptibility to human allogeneic Vγ9Vδ2 T cells.

Allogeneic innate lymphocytes such as Vγ9Vδ2 T cells are attractive candidates for cancer immunotherapy as they provide MHC-unrestricted antitumor activity without clinical evidence for inducing graft-versus-host disease (GvHD). However, current cellular immunotherapy approaches lack predictive biomarkers identifying patient cohorts most susceptible to immune attack. For this purpose we performed a comprehensive analysis of clinical, genetic, metabolic, and immunophenotypic features of 19 primary acute myeloid leukemia (AML) samples and correlated these factors with AML blast recognition by allogeneic Vγ9Vδ2 T cells. We show that 36% of primary AML samples were intrinsically susceptible to allogeneic Vγ9Vδ2 T cells. Among several evaluated features, only UL-16 binding protein 1 (ULBP1) expression (P<0.01) determines intrinsic AML susceptibility to allogeneic Vγ9Vδ2 T cells. Within the intrinsically resistant AML samples, pretreatment of AML blasts with nitrogen-containing bisphosphonates (NBP) significantly induced Vγ9Vδ2 T-cell cytotoxicity in 50% of AML samples, whereas 50% of AML samples were consistently refractory to γδ T-cell cytolysis. Activity of the mevalonate pathway (P<0.05) and myelomonocytic differentiation of AML (P<0.05) correlated with sensitivity of primary AML samples toward NBP pretreatment. In conclusion, this study identifies subsets of AML patients most likely to benefit from allogeneic Vγ9Vδ2 T-cell-mediated immunotherapy.

Specific inhibitory effects of the NO donor MAHMA/NONOate on human platelets.

Nitric oxide (NO) is a physiological inhibitor of platelet function and has vaso-dilating effects. Therefore, synthesized NO releasing agents are used e.g. in cardiovascular medicine. The aim of this study was to characterise specific effects of the short living agent MAHMA/NONOate, a NO donor of the diazeniumdiolate class, on human platelets. Whole blood was obtained from healthy volunteers. In washed human platelets, the MAHMA/NONOate induced phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) and cyclic nucleotide production were studied by Western Blot and by enzyme immunoassay kits. Agonist induced aggregation was measured in platelet rich plasma. Paired Student׳s t-test was used for statistical analysis. MAHMA/NONOate significantly stimulated platelet VASP phosphorylation in a concentration dependent manner and increased intracellular cGMP, but not cAMP levels, transiently. ODQ, a specific inhibitor of the soluble guanylyl cyclase, completely prevented VASP phosphorylation induced by low MAHMA/NONOate concentrations (5nM-15nM). The effects of higher concentrations (30-200nM) were only partially inhibited by ODQ. MAHMA/NONOate reduced platelet aggregation induced by low doses of agonists (2µM ADP, 0.5µg/mL collagen, 5µM TRAP-6) in a concentration dependent manner. MAHMA/NONOate leads to a rapid and transient activation of platelet inhibitory systems, accompanied by decreased platelet aggregation induced by low dose agonists. At low MAHMA/NONOate concentrations, the effects are cGMP dependent and at higher concentrations additionally cGMP independent. The substance could be of interest for clinical situations requiring transient and subtotal inhibition of platelet function.

Increasing susceptibility of nitric oxide-mediated inhibitory platelet signaling during storage of apheresis-derived platelet concentrates.

BACKGROUND: Storage of platelets (PLTs) affects PLT integrity and functionality, a process named the PLT storage lesion. Normal PLT function essentially depends on the balanced interaction of activating and inhibitory signaling pathways. As there are poor data on the alterations of inhibitory signaling during storage of PLT concentrates, this study investigates the modulation capability of the cyclic nucleotide-mediated inhibitory pathways by use of the nitric oxide donor diethylamine diazenium diolate (DEA/NO). STUDY DESIGN AND METHODS: PLTs were obtained from whole blood (WB) and from apheresis-derived PLT concentrates (APCs) stored for 0, 2, and 5 days. Vasodilator-stimulated phosphoprotein (VASP) phosphorylation, cyclic nucleotide concentrations, fibrinogen binding, and agonist-induced aggregation were measured without or after stimulation with DEA/NO. RESULTS: DEA/NO-induced VASP phosphorylation was significantly higher in PLTs from APCs on Days 2 and 5 compared to WB, conditioned by a stronger increase of cyclic guanosine monophosphate (cGMP), but not cyclic adenosine monophosphate (cAMP), in stored PLTs. A quantity of 5 nmol/L DEA/NO neither influenced thrombin receptor activator peptide 6 and collagen-induced aggregation nor fibrinogen binding in freshly collected PLTs, whereas it significantly inhibited both in stored PLTs. CONCLUSION: Stored PLTs showed an impairment of intracellular cGMP regulation, resulting in exceeding inhibition of agonist-induced aggregation and fibrinogen binding in the course of storage. The observed effects could be an important mechanism contributing to the storage lesion with reduced activating potential of PLTs.

Late-onset myasthenia gravis - CTLA4(low) genotype association and low-for-age thymic output of naïve T cells.

Late-onset myasthenia gravis (LOMG) has become the largest MG subgroup, but the underlying pathogenetic mechanisms remain mysterious. Among the few etiological clues are the almost unique serologic parallels between LOMG and thymoma-associated MG (TAMG), notably autoantibodies against acetylcholine receptors, titin, ryanodine receptor, type I interferons or IL-12. This is why we checked LOMG patients for two further peculiar features of TAMG - its associations with the CTLA4(high/gain-of-function) +49A/A genotype and with increased thymic export of naïve T cells into the blood, possibly after defective negative selection in AIRE-deficient thymomas. We analyzed genomic DNA from 116 Caucasian LOMG patients for CTLA4 alleles by PCR/restriction fragment length polymorphism, and blood mononuclear cells for recent thymic emigrants by quantitative PCR for T cell receptor excision circles. In sharp contrast with TAMG, we now find that: i) CTLA4(low) +49G(+) genotypes were more frequent (p = 0.0029) among the 69 LOMG patients with age at onset ≥60 years compared with 172 healthy controls; ii) thymic export of naïve T cells from the non-neoplastic thymuses of 36 LOMG patients was lower (p = 0.0058) at diagnosis than in 77 age-matched controls. These new findings are important because they suggest distinct initiating mechanisms in TAMG and LOMG and hint at aberrant immuno-regulation in the periphery in LOMG. We therefore propose alternate defects in central thymic or peripheral tolerance induction in TAMG and LOMG converging on similar final outcomes. In addition, our data support a 60-year-threshold for onset of 'true LOMG' and an LOMG/early-onset MG overlapping group of patients between 40 and 60.

Five donors-one recipient: modeling a mosaic of granulocytes, natural killer and T cells from cord-blood and third-party donors.

BACKGROUND: A 21-year-old man was admitted to hospital because of leukocytosis, thrombocytopenia and anemia. The patient had been in good health until a few days earlier, when he developed fever and night sweats and his performance status dramatically declined. INVESTIGATIONS: Laboratory tests, immunophenotyping, cytogenetic analyses, bone-marrow biopsy, minimal residual disease analysis using quantitative real-time polymerase chain reaction, differential chimerism analysis using flow cytometry, mixed chimerism analysis, CT scans, electro-encephalography, cerebral magnetic resonance tomography. DIAGNOSIS: Bcr-abl-positive and Philadelphia-chromosome-positive acute lymphoblastic leukemia, and primary graft failure complicated by invasive fungal infection and cytomegalovirus encephalitis. MANAGEMENT: Double cord-blood rescue transplantation, third-party CD34-positive stem-cell rescue transplantation, third-party cytomegalovirus-specific T lymphocyte transplantation.

Whole-genome analysis and HLA genotyping of enteropathy-type T-cell lymphoma reveals 2 distinct lymphoma subtypes.

BACKGROUND & AIMS: Enteropathy-type T-cell lymphoma (ETL) is an aggressive extranodal T-cell non-Hodgkin lymphoma assumed to arise in the setting of celiac disease. METHODS: To precisely define the genetic alterations underlying the pathogenesis of ETL, 30 ETL samples were profiled for genetic copy number alterations using high-resolution whole-genome tiling path array comparative genomic hybridization. To investigate the potential association of genetic alterations in ETL with celiac disease, HLA-DQB1 genotyping was performed. RESULTS: By array comparative genomic hybridization, 13 novel recurrent minimal regions of chromosomal alteration were identified on multiple chromosome arms. ETL is characterized by frequent complex gains of 9q31.3-qter (70% of cases), or by an almost mutually exclusive 2.5-megabase loss of 16q12.1 (23% of cases). Two distinct groups of ETL could be delineated morphologically and genetically: type 1 ETL is characterized by nonmonomorphic cytomorphology, CD56 negativity, and chromosomal gains of 1q and 5q. Type 1 ETL also appears to be linked pathogenetically to celiac disease, sharing genetic alterations and HLA-DQB1 genotype patterns with (refractory) celiac disease. Type 2 ETL shows monomorphic small- to medium-sized tumor cell morphology, frequently shows CD56 expression, MYC oncogene locus gain, and rare gains of chromosomes 1q and 5q. In contrast to type 1 ETL, type 2 ETL shows a HLA-DQB1 genotype pattern more resembling that of the normal Caucasian population. CONCLUSIONS: Contrary to current clinical classification, ETL comprises 2 morphologically, clinically, and genetically distinct lymphoma entities. In addition, type 2 ETL may not be associated with celiac disease.

Frequency analysis of HLA-B7-restricted Epstein-Barr virus-specific cytotoxic T lymphocytes in patients with multiple sclerosis and healthy controls.

The Epstein-Barr virus (EBV) has been implicated in the pathogenesis of multiple sclerosis (MS), however, the mechanisms by which EBV may be involved in MS are unknown. We here have investigated the frequency of EBV-specific cytotoxic T lymphocytes (CTL) in human leukocyte antigen (HLA)-B7(+) patients with MS and healthy controls using enzyme-linked immunospot assays and seven previously characterized HLA-B7-restricted immunogenic EBV peptides. Overall, there were no significant differences in the frequency of EBV-specific CTL between both groups. These data do not support the hypothesis that EBV could play a role in MS by inducing quantitatively altered EBV-specific CTL responses. Other pathogenic mechanisms for EBV in MS remain to be elucidated.

A CTLA4high genotype is associated with myasthenia gravis in thymoma patients.

Myasthenia gravis (MG) in thymoma patients depends critically on intratumorous generation and export of mature autoreactive CD4+ T cells. Why non-MG thymomas fail to produce CD4+ T cells is unknown. We studied three single-nucleotide polymorphisms of the cytotoxic T-lymphocyte-associated antigen 4(CTLA4) gene in thymoma patients, nonthymoma early-onset MG patients, and control subjects. Surprisingly, the CTLA4high genotype +49A/A, which is protective against several autoimmune diseases, exerted a prominent predisposing effect to paraneoplastic MG in thymoma patients. The unusual disease association with a CTLA4high genotype implies a unique pathogenesis of paraneoplastic MG, with high CTLA4 levels possibly supporting the nontolerogenic selection of CD4+ T cells in MG-associated thymomas.