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Flow cytometric immunophenotyping is essential for the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). Most cases have easy to interpret flow cytometry profiles with red cells, neutrophils and monocytes showing complete deficiency of glycophosphatidylinositol (GPI) linked antigen expression. Some cases are more challenging to interpret due to the presence of multiple populations of PNH cells and variable levels of GPI antigen expression.We studied 46 known PNH patients, many with complex immunophenotypic profiles using a novel, single tube, multi-parameter 7-color immunophenotyping assay that allowed simultaneous detection and assessment of PNH clones within multiple lineages of peripheral blood leucocytes. Red cell PNH clones were also assessed in total and immature (CD71+) components by CD59 expression.For individual patients, total PNH clones in each cell lineage were highly correlated. Monocytes, eosinophils and basophils showed the highest proportions of PNH cells. Red cell PNH clones were typically smaller than monocyte and neutrophil PNH clones. In most cases, PNH clones were detectable in minor leucocyte populations where multiple populations of PNH cells were present, variability in the proportions of type II and type III cells was seen across different cell lineages, even though total PNH clones remained similar.This study shows that PNH patients with multiple PNH clones do not always display the same abnormality across all cell lineages routinely tested. There is no simple explanation for this but is likely due to a combination of complex molecular, genetic and biochemical dysfunction in different blood cell types.

Supplementary Figure from Neoadjuvant Intravenous Oncolytic Vaccinia Virus Therapy Promotes Anticancer Immunity in Patients

Supplementary Data from Neoadjuvant Intravenous Oncolytic Vaccinia Virus Therapy Promotes Anticancer Immunity in Patients

Improving the chances of curing patients with cancer who have had surgery to remove metastatic sites of disease is a priority area for cancer research. Pexa-Vec (Pexastimogene Devacirepvec; JX-594, TG6006) is a principally immunotherapeutic oncolytic virus that has reached late-phase clinical trials. We report the results of a single-center, nonrandomized biological end point study (trial registration: EudraCT number 2012-000704-15), which builds on the success of the presurgical intravenous delivery of oncolytic viruses to tumors. Nine patients with either colorectal cancer liver metastases or metastatic melanoma were treated with a single intravenous infusion of Pexa-Vec ahead of planned surgical resection of the metastases. Grade 3 and 4 Pexa-Vec–associated side effects were lymphopaenia and neutropaenia. Pexa-Vec was peripherally carried in plasma and was not associated with peripheral blood mononuclear cells. Upon surgical resection, Pexa-Vec was found in the majority of analyzed tumors. Pexa-Vec therapy associated with IFNα secretion, chemokine induction, and resulted in transient innate and long-lived adaptive anticancer immunity. In the 2 patients with significant and complete tumor necrosis, a reduction in the peripheral T-cell receptor diversity was observed at the time of surgery. These results support the development of presurgical oncolytic vaccinia virus-based therapies to stimulate anticancer immunity and increase the chances to cure patients with cancer.

Improving the chances of curing patients with cancer who have had surgery to remove metastatic sites of disease is a priority area for cancer research. Pexa-Vec (Pexastimogene Devacirepvec; JX-594, TG6006) is a principally immunotherapeutic oncolytic virus that has reached late-phase clinical trials. We report the results of a single-center, nonrandomized biological end point study (trial registration: EudraCT number 2012-000704-15), which builds on the success of the presurgical intravenous delivery of oncolytic viruses to tumors. Nine patients with either colorectal cancer liver metastases or metastatic melanoma were treated with a single intravenous infusion of Pexa-Vec ahead of planned surgical resection of the metastases. Grade 3 and 4 Pexa-Vec–associated side effects were lymphopaenia and neutropaenia. Pexa-Vec was peripherally carried in plasma and was not associated with peripheral blood mononuclear cells. Upon surgical resection, Pexa-Vec was found in the majority of analyzed tumors. Pexa-Vec therapy associated with IFNα secretion, chemokine induction, and resulted in transient innate and long-lived adaptive anticancer immunity. In the 2 patients with significant and complete tumor necrosis, a reduction in the peripheral T-cell receptor diversity was observed at the time of surgery. These results support the development of presurgical oncolytic vaccinia virus-based therapies to stimulate anticancer immunity and increase the chances to cure patients with cancer.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematological disorder, characterized by complement-mediated intravascular hemolysis and thrombosis. The increased incidence of PNH-driven thrombosis is still poorly understood, but unlike other thrombotic disorders, is thought to largely occur through complement-mediated mechanisms. Treatment with a C5 inhibitor, eculizumab, has been shown to significantly reduce the number of thromboembolic events in these patients. Based on previously described links between changes in fibrin clot structure and thrombosis in other disorders, our aim was to investigate clot structure as a possible mechanism of thrombosis in patients with PNH and the anti-thrombotic effects of eculizumab treatment on clot structure. Clot structure, fibrinogen levels and thrombin generation were examined in plasma samples from 82 patients from the National PNH Service in Leeds, UK. Untreated PNH patients were found to have increased levels of fibrinogen and thrombin generation, with subsequent prothrombotic changes in clot structure. No link was found between increasing disease severity and fibrinogen levels, thrombin generation, clot formation or structure. However, eculizumab treated patients showed decreased fibrinogen levels, thrombin generation and clot density, with increasing time spent on treatment augmenting these antithrombotic effects. These data suggest that PNH patients have a prothrombotic clot phenotype due to increased fibrinogen levels and thrombin generation, and that the antithrombotic effects of eculizumab are, in-part, due to reductions in fibrinogen and thrombin generation with downstream effects on clot structure.

Entheseal spinal inflammation and new bone formation with progressive ankylosis may occur in ankylosing spondylitis (AS) and psoriatic arthritis (PsA). This study evaluated whether JAK inhibition with tofacitinib modulated the key disease associated cytokines, TNF and IL-17A and whether tofacitinib also modulated bone marrow stromal cell-derived mesenchymal stem cells (MSCs) function including osteogenesis, since post inflammation new bone formation occurs in these conditions. Methods: Conventional entheseal derived αβ CD4+ and CD8+ T-cells were in-vestigated following anti-CD3/CD28 bead stimulation to determine IL-17A and TNF levels in Tofacitinib treated (1000nM) peri-entheseal bone (PEB) and peripheral blood mononuclear cells (PBMC) following ELISA. Bone marrow stromal cell-derived mesenchymal stem cells (MSCs) colony forming unit (CFU-F) and multilineage potential was evaluated using tofacitinib (dosag-es ranging between 100, 500, 1000 and 10000nM). Results: Induced IL-17A and TNF cytokine production from both entheseal CD4+ T-cells and CD8+ T-cells were effectively inhibited by to-facitinib. Tofacitinib treatment did not impact on CFU-F potential or in vitro chondro- and oste-ogenesis. However, tofacitinib stimulation increased MSC adipogenic potential with greater Oil Red O stained area. Conclusion: Inducible IL-17A and TNF production by healthy human enthe-seal CD4+ and CD8+ T-cells was robustly inhibited in vitro by tofacitinib. However, tofacitinib did not impact on MSC osteogenesis but stimulated in vitro MSC adipogenesis, the rele-vance of which needs further evaluation given the adipocytes are associated with new bone formation in SpA.

Objective To assess the prevalence of the MYD88 L265P mutation and variants within NLRP3 and evaluate the status of oligoclonal hematopoiesis in 30 patients with Schnitzler syndrome (SchS). Methods Thirty patients with SchS were recruited from 3 clinical centers. Six patients with known acquired cryopyrin-associated periodic syndromes (aCAPS) were included as controls. Allele-specific oligonucleotide-polymerase chain reaction was used for the detection of the MYD88 L265P variant, next-generation sequencing was applied to analyze NLRP3 and 28 genes associated with myelodysplastic syndrome, and gene scanning was performed for the detection of X chromosome inactivation. Results Activating NLRP3 mutations were not present in 11 SchS patients who had not been sequenced for this gene previously. The MYD88 L265P variant was present in 9 of 30 SchS patients, and somatic mutations associated with clonal hematopoiesis were identified in 1 of 30 patients with SchS and 1 of 6 patients with aCAPS. Evidence of nonrandom X chromosome inactivation was detected in 1 female patient with SchS and 1 female patient with aCAPS. Conclusion A shared molecular mechanism accounting for the pathogenesis of inflammation in SchS remains elusive. Clonal hematopoiesis is not associated with other somatic mutations found in individuals with SchS or aCAPS.

The IL-23/IL-17 cytokine axis is related to spondyloarthropathy (SpA) pattern diseases that target the skin, eye, gut and joints. These share overlapping target tissues with Th2 type or allergic diseases, including the skin, eye and gut but SpA diseases exhibit distinct microanatomical topography, molecular characteristics, and clinical features including uveitis, psoriasis, apical pulmonary involvement, lower gastrointestinal involvement with colitis, and related arthritides including psoriatic arthritis and ankylosing spondylitis. Inflammatory arthritis is conspicuously absent from the Th2 diseases which are characterised IL-4/IL-13 dependent pathway activation including allergic rhino-conjunctivitis, atopic eczema, allergic asthma and food allergies. This traditional understanding of non-overlap of musculoskeletal territory between that atopic diseases and the IL-17 -mediated SpA diseases is undergoing a critical reappraisal with the recent demonstration of IL-4/IL-13 blockade, may be associated with the development of SpA pattern arthritis, psoriasiform skin disease and occasional anterior uveitis. Given the known plasticity within Th paradigm pathways, these findings suggest dynamic Th2 cytokine and Th17 cytokine counter regulation in vivo in humans. Unexpected, this is the case in peripheral enthesis and when the IL-4/13 immunological brake on IL-23/17 cytokines is removed, a SpA phenotype may emerge. We discuss hitherto unexpected observations in SpA, showing counter regulation between the Th17 and Th2 pathways at sites including the entheses that collectively indicate that the emergent reverse translational therapeutic data is more than coincidental and offers new insights into the “Th paradigms” in atopy and SpA.

Objectives A retrospective population-based study to determine the incidence and prevalence of patients with the rare blood disease paroxysmal nocturnal haemoglobinuria (PNH). Methods All patients were identified by flow cytometric detection of blood cells deficient in glycosylphosphatidylinositol (GPI) linked proteins at a single diagnostic reference laboratory that serves the Yorkshire based, Haematological Malignancy Research Network (HMRN) with a population of 3.8 million. Results One hundred and ninety-seven patients with detectable PNH clones at a level of >0.01% in at least two lineages of cells (neutrophils, monocytes and/or red cells) were identified over a 15-year period (2004-2018). Of these, 88% had aplastic anaemia (AA), 8% classical PNH and 3% myelodysplastic syndrome. The overall incidence rate was estimated at 0.35 cases per 100 000 people per year. This equates to 220 cases newly diagnosed in the United Kingdom each year. The overall prevalence rate was 3.81 per 100 000, this equates to an estimated 2400 prevalent cases in the UK. The overall and relative 5-year survival rates were 72% and 82.7%, respectively. Conclusions This study showed that classical haemolytic PNH is a rare disease and represents only a small proportion overall of patients with detectable PNH cells, the majority of which have aplastic anaemia.

Advancing age and chronic health conditions, significant risk factors for severe COVID-19, are associated with a pro-inflammatory state, termed inflamm-aging. CXCR6+T cells are known to traffic to the lung and have been reported to increase with age. The ligand of CXCR6, CXCL16, is constitutively expressed in the lung and upregulated during inflammatory responses and the CXCR6/CXCL16 axis is associated with severe lung disease and pneumonia. Genome-wide association studies have also recently identified 3p21.31, encompassing theCXCR6gene, as a susceptibility locus for severe COVID-19. We assessed numbers T cells expressing the chemokine receptor CXCR6 and plasma levels of CXCL16, in control and COVID-19 patients. Results demonstrated that circulating CD8+CXCR6+T cells were significantly elevated with advancing age, yet virtually absent in patients with severe COVID-19. Peripheral levels of CXCL16 were significantly upregulated in severe COVID-19 patients compared to either mild COVID-19 patients or SARS-CoV-2 negative controls. This study supports a significant role of the CXCR6/CXCL16 axis in the immunopathogenesis of severe COVID-19.

BackgroundThe Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib provided a breakthrough in the treatment of chronic lymphocytic leukaemia (CLL), but cases of resistance are now emerging. Whilst resistance is commonly associated with mutations in BTK itself and the downstream signalling molecule PLCg2, this is not always the case. In a recent study, we have detected some epigenomic plasticity correlated with the dynamics of CLL cell response to ibrutinib. To understand the mechanisms mediating resistance in CLL, it is important to be able to determine whether the observed phenotypic changes are driven solely by resistance cues (e.g. clonal evolution, activation of signalling bypassing BTK inhibition), or if they could also be the consequence of unrelated events.MethodsTo answer to this question, we have monitored chromatin changes happening in response to ibrutinib from the start of treatment until relapse in CLL cells from both a patient carrying a previously identified BTK C481S mutation and from a relapsing patient, for whom none of the classical genetic lesions associated with ibrutinib relapse was detected.ResultsWe established that the epigenome and gene expression in CLL cells from patients on ibrutinib changes with time independently of disease progression and identified two patterns of chromatin alterations, which are independent of resistance mechanisms; one ibrutinib-dependent and the other related to quiescence. Furthermore, by defining the main characteristics of resistance-independent epigenomic plasticity and excluding them from the analysis, we identified candidate genes potentially mediating disease progression.ConclusionThis two-step strategy could fundamentally alter the understanding of resistance to treatment in CLL.

BackgroundThe human enthesis conventional T cells are poorly characterised.ObjectivesTo study the biology of the conventional T cells in human enthesis.MethodsCD4+ and CD8+ T cells were investigated in 25 enthesis samples using immunofluorescence, cytometrically, bulk RNAseq and quantitative real-time PCR following anti-CD3/CD28 bead stimulation to determine interleukin (IL)-17A and tumour necrosis factor (TNF) levels. T-cell receptor (TCR) repertoires were characterised and a search for putative T-cell reactivity was carried out using TCR3 database. The impact of pharmacological antagonism with retinoic acid receptor-related orphan nuclear receptor gamma t inhibitor (RORγti), methotrexate and phosphodiesterase type 4 inhibitor (PDE4i) was investigated.ResultsImmunofluorescence and cytometry suggested entheseal resident CD4+ and CD8+ T cells with a resident memory phenotype (CD69+/CD45RA-) and tissue residency gene transcripts (higher NR4A1/AhR and lower KLF2/T-bet transcripts). Both CD4+ and CD8+ T cells showed increased expression of immunomodulatory genes including IL-10 and TGF-β compared with peripheral blood T cells with entheseal CD8+ T cells having higher CD103, CD49a and lower SIPR1 transcript that matched CD4+ T cells. Following stimulation, CD4+ T cells produced more TNF than CD8+ T cells and IL-17A was produced exclusively by CD4+ T cells. RNAseq suggested both Cytomegalovirus and influenza A virus entheseal resident T-cell clonotype reactivity. TNF and IL-17A production from CD4+ T cells was effectively inhibited by PDE4i, while RORγti only reduced IL-17A secretion.ConclusionsHealthy human entheseal CD4+ and CD8+ T cells exhibit regulatory characteristics and are predicted to exhibit antiviral reactivity with CD8+ T cells expressing higher levels of transcripts suggestive of tissue residency. Inducible IL-17A and TNF production can be robustly inhibited in vitro.

Introduction La place centrale de l’IL-17A dans la physiopathologie de la spondyloarthrite axiale (AxSpA) a ete confirmee par le succes des anti-IL-17A. Cependant, la nature des populations cellulaires produisant cette importante cytokine pro-inflammatoire reste mal definie. Nous avons donc caracterise les principales populations cellulaires productrices d’IL-17A dans la AxSpA, et plus particulierement une population de lymphocytes T non conventionnels, les MAIT (Mucosal Associated Invariant T cells). Patients et methodes La production d’IL-17A de 5 populations de cellules sanguines peripheriques triees, a savoir les MAIT, les lymphocytes Tγ δ, les T CD4+, les T CD8+ et les neutrophiles a ete evaluee avant et apres stimulation par l’association de PMA, d’un ionophore calcique A23187 et de la B1,3 glucane. L’expression genique et proteique d’IL-17A etait respectivement determinee par la technologie nCounter et la technologie ultra-sensible SimoA. Les MAIT provenant d’entheses axiales de temoins sains (n = 5) etaient caracterises par cytometrie en flux, qPCR et leur production d’IL-17 evaluee apres stimulation. Resultats Dans le sang, a l’echelle cellulaire, les MAIT produisaient la plus grande quantite d’IL-17A par rapport aux T CD4+ (p Conclusion Les MAIT circulants et les MAIT residents dans les entheses axiales contribuent a la production d’IL-17 et pourraient jouer un role important dans la physiopathologie de l’AxSpA.

Significant challenges exist to develop sensitive and specific tests to diagnose COVID-19 caused by infection with the RNA virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).1 Diagnosis of COVID-19 in the acute stage relies on detecting SARS-CoV-2 genomic RNA in naso/oropharyngeal (NOP) swabs. The swab is transferred to the laboratory in either viral or universal transport medium (VTM/UTM) and reverse transcriptase PCR (RT-PCR) performed to detect SARS-CoV-2 RNA.1 This same algorithm is used in the diagnosis of seasonal flu caused by the RNA virus, influenza with few false-positive and false-negative results leading to specificity and sensitivity greater than 95%, respectively.2 The false-negative rate for RT-PCR in COVID-19 has been reported to be as high as 41%3 and there are several reports of swab negative patients, who are subsequently positive on repeat testing.4 It is possible that the amount of virion present in NOP in COVID-19 is much less than that in influenza …

A retrospective analysis of presentation clinical, laboratory and immunophenotypic features of 1 081 patients with paroxysmal nocturnal haemoglobinuria (PNH) clones [glycosylphosphatidylinositol (GPI)‐deficient blood cells] identified at our hospital by flow cytometry over the past 25 years was undertaken. Three distinct clusters of patients were identified and significant correlations between presentation disease type and PNH clone sizes were evident. Smaller PNH clones predominate in cytopenic and myelodysplastic subtypes; large PNH clones were associated with haemolytic, thrombotic and haemolytic/thrombotic subtypes. Rare cases with an associated chronic myeloproliferative disorder had either large or small PNH clones. Cytopenia was a frequent finding, highlighting bone marrow failure as the major underlying feature associated with the detection of PNH clones in the peripheral blood. Red cell PNH clones showed significant correlations between the presence of type II (partial GPI deficiency) red cells and thrombotic disease. Haemolytic PNH was associated with type III (complete GPI deficiency) red cell populations of >20%. Those with both haemolytic and thrombotic features had major type II and type III red cell populations. Distinct patterns of presentation age decade were evident for clinical subtypes with a peak incidence of haemolytic PNH in the 30–49 year age group and a biphasic age distribution for the cytopenia group.

ObjectivesMurine models of interleukin (IL)-23-driven spondyloarthritis (SpA) have demonstrated entheseal accumulation of γδT-cells which were responsible for the majority of local IL-17A production. However, IL-23 blockers are ineffective in axial inflammation in man. This study investigated γδT-cell subsets in the normal human enthesis to explore the biology of the IL-23/17 axis.MethodsHuman spinous processes entheseal soft tissue (EST) and peri-entheseal bone (PEB) were harvested during elective orthopaedic procedures. Entheseal γδT-cells were evaluated using immunohistochemistry and isolated and characterised using flow cytometry. RNA was isolated from γδT-cell subsets and analysed by qPCR. Entheseal γδT-cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, anti-CD3/28 or IL-23 and IL-17A production was measured by high-sensitivity ELISA and qPCR.ResultsEntheseal γδT-cells were confirmed immunohistochemically with Vδ1 and Vδ2 subsets that are cytometrically defined. Transcript profiles of both cell populations suggested tissue residency and immunomodulatory status. Entheseal Vδ2 cells expressed high relative abundance of IL-23/17-associated transcripts including IL-23R, RORC and CCR6, whereas the Vδ1 subset almost completely lacked detectable IL-23R transcript. Following PMA stimulation IL-17A was detectable in both Vδ1 and Vδ2 subsets, and following CD3/CD28 stimulation both subsets showed IL-17A and IL-17F transcripts with neither transcript being detectable in the Vδ1 subset following IL-23 stimulation.ConclusionSpinal entheseal Vδ1 and Vδ2 subsets are tissue resident cells with inducible IL-17A production with evidence that the Vδ1 subset does so independently of IL-23R expression.

To assess the prevalence of the MYD88 L265P mutation and variants within NLRP3 and evaluate the status of oligoclonal hematopoiesis in 30 patients with Schnitzler syndrome (SchS).Thirty patients with SchS were recruited from 3 clinical centers. Six patients with known acquired cryopyrin-associated periodic syndromes (aCAPS) were included as controls. Allele-specific oligonucleotide-polymerase chain reaction was used for the detection of the MYD88 L265P variant, next-generation sequencing was applied to analyze NLRP3 and 28 genes associated with myelodysplastic syndrome, and gene scanning was performed for the detection of X chromosome inactivation.Activating NLRP3 mutations were not present in 11 SchS patients who had not been sequenced for this gene previously. The MYD88 L265P variant was present in 9 of 30 SchS patients, and somatic mutations associated with clonal hematopoiesis were identified in 1 of 30 patients with SchS and 1 of 6 patients with aCAPS. Evidence of nonrandom X chromosome inactivation was detected in 1 female patient with SchS and 1 female patient with aCAPS.A shared molecular mechanism accounting for the pathogenesis of inflammation in SchS remains elusive. Clonal hematopoiesis is not associated with other somatic mutations found in individuals with SchS or aCAPS.

We thank Alunno et al 1 for their comments on our paper reporting on the isolation and preliminary characterisation of normal human enthesis CD4+ and CD8+ conventional T-cells.2 Our work showed that up to 2% of CD4+ T cells were Th17 in nature as demonstrated by intracellular flow cytometry. We were unable to identify FOXP3+ Tregs at the enthesis, but these were readily demonstrable in peripheral blood from the same patients. Alunno and colleagues1 point towards the transdifferentiation between Th17s and Tregs that may occur in vivo and which can be modulated by pivotal spondyloarthritis (SpA) associated cytokines including TNF. Our results were generated in an artificial ‘in vitro enthesitis’ model, and we agree with the comments by Alunno et al 1 in regard to the role …

Background:We have previously reported that the normal spinal enthesis has populations of conventional T-cells including CD4+ & CD8+ T-cells that could be induced to produce IL-17A and TNF following anti-CD3/CD28 stimulation. The biology of such cells in health including their normal function and antigen reactivity is completely unknown. The purpose of this work was to define the phenotype, functionality and TCR reactivity of such T-cells in health.Objectives:To investigate whether the T-cells at the normal enthesis were regulatory in nature and to determine the type of regulatory T-cell as Tr1 or FOXP3 regulatory T-cell and to determine T-cell reactivity.Methods:Healthy interspinous ligament and spinous process with matched peripheral blood were harvested from patients undergoing elective spinal surgery (n=20). Entheseal soft tissue (EST) & peri-entheseal bone (PEB) was enzymatically digested and then sorted. Tr1 and Treg phenotypes were investigated using flow cytometry. Analysis of cytokines, growth factors and chemokines was performed by qRT-PCR, ELISA and flow cytometry. TCR sequencing was performed and a search for putative T-cell reactivity was done using TCR3 database.Results:Pro-inflammatory cytokine transcripts including IL-17A, IL-17F, IL-22, IL-23 (p19) & TNF were very low or undetectable in the Enthesis T-cells (Fig 1). Flow cytometry confirmed entheseal T-cells had a Tr1 phenotype (CD4+ LAG3+ CD49b+). Intracellular flow cytometry showed enthesis T-cells had very low FOXP3 expression, when compared to their blood counterparts. Intracellular flow cytometry and gene expression showed high basal expression of growth factors and regulatory proteins such as IL-10 & TGFβ, when compared to blood T-cells. RNA-Seq data, showed 13 potential TCR clonal sequences the most common of which are predicted to be reactive viral infection was CMV present in 8 sequences and Influenza A virus present in 2 sequences.Conclusion:The healthy human enthesis has regulatory T-cells of a Tr1 phenotype rather than a FOXP3 Treg phenotype. Many clones have antigen specificity indicating reactivity to prior infection. These findings suggest that conventional entheseal T-cells have a role in enthesis immune homeostasis.Disclosure of Interests:Hannah Rowe Grant/research support from: Novartis UK Investigator Initiated non-clinical research funding support, Abdulla Watad: None declared, Tobias Russell Grant/research support from: Novartis UK Investigator Initiated non-clinical research funding support, Kassem Sharif: None declared, Darren Newton: None declared, Miriam Wittmann: None declared, Qiao Zhou Grant/research support from: Funded by the PARTNER fellowship program, Almas Khan: None declared, Peter Loughenbury: None declared, Robert Dunsmuir: None declared, Abhay S Rao: None declared, Peter Millner: None declared, Tony Kenna: None declared, Matthew Brown: None declared, Charlie Bridgewood: None declared, Dennis McGonagle Grant/research support from: Janssen Research & Development, LLC

Paroxysmal nocturnal hemoglobinuria is a rare acquired hematologic disorder, the most serious complication of which is thrombosis. The increased incidence of thrombosis in paroxysmal nocturnal hemoglobinuria is still poorly understood, but unlike many other thrombotic disorders, predominantly involves complement-mediated mechanisms. This review article discusses the different factors that contribute to the increased risk of thrombosis in paroxysmal nocturnal hemoglobinuria. Paroxysmal nocturnal hemoglobinuria leads to a complex and multifaceted prothrombotic state due to the pathological effects of platelet activation, intravascular hemolysis and neutrophil/monocyte activation. Platelet and endothelial microparticles as well as oxidative stress may play a role. Impaired fibrinolysis has also been observed and may be caused by several mechanisms involving interactions between complement activation, coagulation and fibrinolysis. While many factors may affect thrombosis in paroxysmal nocturnal hemoglobinuria, the relative contribution of each mechanism that has been implicated is difficult to quantify. Further studies, including novel in vivo and in vitro thrombosis models, are required in order to define the role of the individual mechanisms contributing to thrombosis, impaired fibrinolysis and clarify other complement-driven prothrombotic mechanisms in paroxysmal nocturnal hemoglobinuria.

Chronic lymphocytic leukaemia (CLL) is the most common clonal B-cell disorder characterized by clonal diversity, a relapsing and remitting course, and in its aggressive forms remains largely incurable. Current front-line regimes include agents such as fludarabine, which act primarily via the DNA damage response pathway. Key to this is the transcription factor p53. Mutations in the TP53 gene, altering p53 functionality, are associated with genetic instability, and are present in aggressive CLL. Furthermore, the emergence of clonal TP53 mutations in relapsed CLL, refractory to DNA-damaging therapy, suggests that accurate detection of sub-clonal TP53 mutations prior to and during treatment may be indicative of early relapse. In this study, we describe a novel deep sequencing workflow using multiple polymerases to generate sequencing libraries (MuPol-Seq), facilitating accurate detection of TP53 mutations at a frequency as low as 0.3%, in presentation CLL cases tested. As these mutations were mostly clustered within the regions of TP53 encoding DNA-binding domains, essential for DNA contact and structural architecture, they are likely to be of prognostic relevance in disease progression. The workflow described here has the potential to be implemented routinely to identify rare mutations across a range of diseases.

Minimal residual disease (MRD) negativity, defined as

Plasma cells (PCs), the terminal effectors of humoral immunity, are short-lived unless supported by niche environments in which they may persist for years. No model system has linked B cell activation with niche function to allow the in vitro generation of long-lived PCs. Thus, the full trajectory of B cell terminal differentiation has yet to be investigated in vitro. In this article, we describe a robust model for the generation of polyclonal long-lived human PCs from peripheral blood B cells. After a proliferative plasmablast phase, PCs persist in the absence of cell division, with viability limited only by elective culture termination. Conservative predictions for PC life expectancy are 300 d, but with the potential for significantly longer life spans for some cells. These long-lived PCs are preferentially derived from memory B cells, and acquire a CD138high phenotype analogous to that of human bone marrow PCs. Analysis of gene expression across the system defines clusters of genes with related dynamics and linked functional characteristics. Importantly, genes in these differentiation clusters demonstrate a similar overall pattern of expression for in vitro and ex vivo PCs. In vitro PCs are fully reprogrammed to a secretory state and are adapted to their secretory load, maintaining IgG secretion of 120 pg/cell/day in the absence of XBP1 mRNA splicing. By establishing a set of conditions sufficient to allow the development and persistence of mature human PCs in vitro, to our knowledge, we provide the first platform with which to sequentially explore and manipulate each stage of human PC differentiation.

Although gammadelta T cells play a role in protecting tissues from pathogen-elicited damage to bacterial, viral and parasitic pathogens, the mechanisms involved in the damage and in the protection have not been clearly elucidated. This has been addressed using a murine model of listeriosis, which in mice lacking gammadelta T cells (TCRdelta(-/-)) is characterised by severe and extensive immune-mediated hepatic necrosis. We show that these hepatic lesions are caused by Listeria-elicited CD8(+) T cells secreting high levels of TNF-alpha that accumulate in the liver of Listeria-infected TCRdelta(-/-) mice. Using isolated populations of gammadelta T cells from wild-type and cytokine-deficient strains of mice to reconstitute TCRdelta(-/-) mice, the TCR variable gene 4 (Vgamma4)(+) subset of gammadelta T cells was shown to protect against liver injury. Hepatoprotection was dependent upon their ability to produce IL-10 after TCR-mediated interactions with Listeria-elicited macrophages and CD8(+) T cells. IL-10-producing Vgamma4(+) T cells also contribute to controlling CD8(+) T cell expansion and to regulating and reducing TNF-alpha secretion by activated CD8(+) T cells. This effect on TNF-alpha production was directly attributed to IL-10. These findings identify a novel mechanism by which pathogen-elicited CD8(+) T cells are regulated via interactions with, and activation of, IL-10-producing hepatoprotective gammadelta T cells.

Although gammadelta T cells are involved in the response to many pathogens, the dynamics and heterogeneity of the local gammadelta T cell response remains poorly defined. We recently identified gammadelta T cells as regulators of macrophages and dendritic cells during the resolution of Streptococcus pneumoniae-mediated lung inflammation. Here, using PCR, spectratype analysis and flow cytometry, we show that multiple gammadelta T cell subsets, including those bearing Vgamma1, Vgamma4 and Vgamma6 TCR, increase in number in the lungs of infected mice, but not in associated lymphoid tissue. These gammadelta T cells displayed signs of activation, as defined by CD69 and CD25 expression. In vivo BrdU incorporation suggested that local expansion, rather than recruitment, was the principal mechanism underlying this increase in gammadelta T cells. This conclusion was supported by the finding that pulmonary gammadelta T cells, but not alphabeta T cells, isolated from mice that had resolved infection exhibited lung-homing capacity in both naive and infected recipients. Together, these data provide novel insights into the origins of the heterogeneous gammadelta T cell response that accompanies lung infection, and the first evidence that inflammation-associated gammadelta T cells may exhibit distinct tissue-homing potential.

To ensure invading pathogens are eliminated with minimal damage to host tissues it is essential that macrophage activation be tightly regulated. Previously we demonstrated that a subset of gammadelta T cells (Vgamma1(+)) contributes to resolving pathogen-induced immune responses by killing activated macrophages. However, the exaggerated macrophage response seen in infected Vgamma1(+) T cell-deficient mice suggests that gammadelta T cells play a broader role in macrophage homeostasis and other subsets might promote macrophage activation. Using a macrophage:gammadelta T cell co-culture system we have shown that gammadelta T cells increase the activity of macrophages activated in vivo by Listeria monocytogenes infection. In a dose-dependent manner, gammadelta T cells up-regulated production of cytokines (TNF-alpha, IL-6, IL-10) and chemokines (MIP-1alpha, MIP-1beta) by Listeria-elicited macrophages. The ability to increase macrophage cytokine production was prominent among Vgamma4(+) gammadelta T cells. Reciprocally, Vgamma4(+) gammadelta T cells were activated by Listeria-elicited macrophages, resulting in production of the anti-inflammatory cytokine, IL-10. gammadelta T cell adoptive transfer experiments showed that Vgamma4(+) T cells protected TCRdelta(-/-) mice against Listeria-induced liver injury and necrosis. These findings identify distinct and non-overlapping roles for gammadelta T cell subsets in regulating macrophage function during pathogen-induced immune responses.

Although γδ T cells are a common feature of many pathogen-induced immune responses, the factors that influence, promote, or regulate the response of individual γδ T-cell subsets to infection is unknown. Here we show that in the absence of Vγ1+T cells, novel subsets of γδ T cells, expressing T-cell receptor (TCR)-Vγ chains that normally define TCRγδ+dendritic epidermal T cells (DETCs) (Vγ5+), intestinal intraepithelial lymphocytes (iIELs) (Vγ7+), and lymphocytes associated with the vaginal epithelia (Vγ6+), are recruited to the spleen in response to bacterial infection in TCR-Vγ1−/−mice. By comparison of phenotype and structure of TCR-Vγ chains and/or -Vδ chains expressed by these novel subsets with those of their epithelium-associated counterparts, the Vγ6+T cells elicited in infected Vγ1−/−mice were shown to be identical to those found in the reproductive tract, from where they are presumably recruited in the absence of Vγ1+T cells. By contrast, Vγ5+and Vγ7+T cells found in infected Vγ1−/−mice were distinct from Vγ5+DETCs and Vγ7+iIELs. Functional analyses of the novel γδ T-cell subsets identified for infected Vγ1−/−mice showed that whereas the Vγ5+and Vγ7+subsets may compensate for the absence of Vγ1+T cells by producing similar cytokines, they do not possess cytocidal activity and they cannot replace the macrophage homeostasis function of Vγ1+T cells. Collectively, these findings identify novel subsets of γδ T cells, the recruitment and activity of which is under the control of Vγ1+T cells.

Gammadelta T cells play important but poorly defined roles in pathogen-induced immune responses and in preventing chronic inflammation and pathology. A major obstacle to defining their function is establishing the degree of functional redundancy and heterogeneity among gammadelta T cells. Using mice deficient in Vgamma1+ T cells which are a major component of the gammadelta T cell response to microbial infection, a specific immunoregulatory role for Vgamma1+ T cells in macrophage and gammadelta T cell homeostasis during infection has been established. By contrast, Vgamma1+ T cells play no significant role in pathogen containment or eradication and cannot protect mice from immune-mediated pathology. Pathogen-elicited Vgamma1+ T cells also display different functional characteristics at different stages of the host response to infection that involves unique and different populations of Vgamma1+ T cells. These findings, therefore, identify distinct and nonoverlapping roles for gammadelta T cell subsets in infection and establish the complexity and adaptability of a single population of gammadelta T cells in the host response to infection that is not predetermined, but is, instead, shaped by environmental factors.

The second to fourth digit ratio (2D:4D) is sexually dimorphic, with lower mean values in males compared to females. It has been suggested that the sex difference in 2D:4D is determined prenatally, 2D:4D is negatively related to prenatal testosterone and positively to prenatal oestrogen, and that 2D:4D is a marker for levels of sex steroids during brain organisation. There is growing evidence that many sex-dependent behaviours are correlated with 2D:4D. However, there is no direct evidence for an effect of prenatal sex steroids on the digit ratio. The response to prenatal testosterone is dependent on the amount produced and the foetal sensitivity to the hormone. Variation in the X-linked androgen receptor gene (AR) determines sensitivity to testosterone. Alleles of AR with low numbers of CAG triplets respond to testosterone with high transactivational activity, while high numbers of CAG's are associated with increased insensitivity to testosterone. We show in a sample of 50 men (49 Caucasian subjects, 1 Caucasian/Chinese subject) that 2D:4D is a phenotypic correlate of AR structure. Right-hand 2D:4D was positively correlated with CAG number and individuals with low 2D:4D in their right hand compared to left hand had AR alleles with low CAG numbers. We discuss the implications of our findings for our understanding of the aetiology of 2D:4D, its relationships with sex-dependent behaviours, and the evolutionary implications of variation in 2D:4D and AR.

PROBLEM: Detailed analysis of the expression of natural killer (NK) cell activatory and inhibitory receptors by human decidual leukocyte subpopulations has not been undertaken. METHOD OF STUDY: Expression of the natural cytotoxicity receptors NKp30, NKp44 and NKp46 by decidual leukocytes were studied by reverse transcriptase polymerase chain reaction. Expression of the killer cell Ig-like receptors CD158a and CD158b on decidual T cells were studied by flow cytometry. RESULTS: First trimester decidual leukocytes expressed mRNA for the NKp30 and NKp46 receptors but expression of NKp44, a marker of activated NK cells, was not detected. A mean of 11.8 and 15.8% of decidual T cells expressed CD158a or 158b, respectively, while only around 1% of peripheral blood T cells were CD158a + or CD158b + . CONCLUSIONS: Like peripheral blood NK cells, decidual NK cells express the natural cytotoxicity receptors NKp30 and NKp46 but the significance of this will not become apparent until ligands for these molecules have been identified. Only a minority of decidual T cells express CD158, indicating that this is not a mechanism for inhibition of cytotoxicity mediated by all decidual T cells.

Chronic lymphocytic leukemia (CLL) -like monoclonal B-cell lymphocytosis (MBL) shares common immunophenotype and cytogenetic abnormalities with CLL, from which it is discriminated by a cutoff value of 5 × 10(9)/L circulating clonal B cells. However, the clonal size in MBL is extremely variable and allows discrimination of two distinct entities (high-count [HC] and low-count [LC]-MBL) based on a cutoff value of 0.5 × 10(9)/L clonal B cells. HC-MBL is associated with lymphocytosis and progresses to CLL requiring treatment at a rate of 1.1% per year, whereas LC-MBL is found in the general population only through high-sensitivity techniques and carries limited, if any, risk of progression. We performed an immunogenetic profiling of 333 cases with CLL-like MBL supplemented by detailed comparisons with CLL, focusing especially on CLL Rai stage 0 (CLL-0). LC- and HC-MBL had similar somatic hypermutation status, yet different IGHV gene repertoires and frequencies of B-cell receptor (BcR) stereotypy. In particular, stereotyped BcRs were infrequent in LC-MBL and were often not CLL specific. In contrast, HC-MBL exhibited clear immunogenetic similarities to CLL-0. These findings indicate that LC-MBL may not represent a true preleukemic condition, thus differing from HC-MBL/CLL-0 in which the identification of factors endowing malignant potential is strongly warranted.

Antimicrobial proteins influence intestinal microbial ecology and limit proliferation of pathogens, yet the regulation of their expression has only been partially elucidated. Here, we have identified a putative pathway involving epithelial cells and intestinal intraepithelial lymphocytes (iIELs) that leads to antimicrobial protein (AMP) production by Paneth cells. Mice lacking γδ iIELs (TCRδ-/-) express significantly reduced levels of the AMP angiogenin 4 (Ang4). These mice were also unable to up-regulate Ang4 production following oral challenge by Salmonella, leading to higher levels of mucosal invasion compared to their wild type counterparts during the first 2 hours post-challenge. The transfer of γδ iIELs from wild type (WT) mice to TCRδ-/- mice restored Ang4 production and Salmonella invasion levels were reduced to those obtained in WT mice. The ability to restore Ang4 production in TCRδ-/- mice was shown to be restricted to γδ iIELs expressing Vγ7-encoded TCRs. Using a novel intestinal crypt co-culture system we identified a putative pathway of Ang4 production initiated by exposure to Salmonella, intestinal commensals or microbial antigens that induced intestinal epithelial cells to produce cytokines including IL-23 in a TLR-mediated manner. Exposure of TCR-Vγ7+ γδ iIELs to IL-23 promoted IL-22 production, which triggered Paneth cells to secrete Ang4. These findings identify a novel role for γδ iIELs in mucosal defence through sensing immediate epithelial cell cytokine responses and influencing AMP production. This in turn can contribute to the maintenance of intestinal microbial homeostasis and epithelial barrier function, and limit pathogen invasion. © 2013 Walker et al.

Introduction Chronic lymphocytic leukemia (CLL) is one of the most common B cell malignancies in the western world. The importance of B cell receptor (BCR) signaling in CLL pathology has led to the emergence of a number of targeted therapies specific to kinases in this pathway. Ibrutinib, a potent irreversible Bruton's Tyrosine Kinase (BTK) inhibitor, is highly effective in CLL with minimal side effects. However, resistance to Ibrutinib is now emerging among patients in several clinical trials. Aims/Methods To understand the molecular mechanisms underlying ibrutinib resistance to aid in early detection of resistant clones and to inform further treatment choices, we have developed an in vitro ibrutinib resistant model using BCR signalling dependent B cell lymphoma lines (Ramos and Namalwa). These cells have been conditioned by exposure to escalating doses of ibrutinib - starting from 0.001μM and increased over time to a lethal dose of 0.5μM in order to select drug-resistant cells. Single cell clones have been isolated from both the naïve and the resistant cells. The growth kinetics of 7 Ramos and 5 Namalwa resistant clones has reached the same pattern as the parental cells. Additionally, 3 naïve clones were isolated from each sensitive parental cell line. Dysregulation of the BCR signaling pathway was characterized by both flow cytometry, using specific anti-phosphokinase antibodies, and by Western blotting. In addition, RNA from these clones was isolated and analyzed on an Affymetrix HTA2.0 gene expression array (n=6 from two cell lines paralleled to one naïve clone from each cell line). Results In Ramos (n=3), gene expression profiling revealed significant upregulation of BCL2 and interferon response genes including interferon gamma (IFNG). However, in Namalwa (n=3), in addition to BCL2 upregulation, the expression arrays showed the overexpression of BCR signaling pathway genes including BTK itself, CD79B, LCK and GAB2 (Growth factor receptor-bound protein 2- associated-binder 2), suggesting hyper-activation of BCR pathway and constitutive activation of BTK and downstream kinases. Moreover, initial screening showed that not all of the resistant clones behave similarly. In Ramos, one clone apparently escapes ibrutinib inhibition by modifying different pathways compared to the other analysed clones, suggesting the potential for multiple mechanisms by which the malignant B cell clones can evade the toxic activity of targeted therapy. Results of this analysis were validated by qPCR and additional resistant clones were tested for BCL2 and IFNG expression. In Ramos, 7 resistant clones showed significant upregulation of BCL2 (mean 9.7-fold increase, range 2.1- 31.8, SEM 4.5) and IFNG(mean increase 8.3, range 2.6-19.1, SEM 2.2). IFNγ, is a known immune modulator linked to various stress signals indicating one possible resistance mechanism of malignant cells to anti-cancer therapies. In Namalwa, qPCR of 5 resistant clones showed significant upregulation of BTK (mean increase 10.3, range 2- 31: SEM: 5.4) and confirmed at the protein expression level by Western blotting as well; where the resistant cells over-express BTK protein compared to their respective naïve cells (n=6, mean increase 1.6, range 1.1- 1.9, SEM 0.16). Moreover, CD79B was over-expressed (mean over-expression 12.4, range 1.5- 22.2, SEM: 4.3) and 6-fold overexpression of LCK (mean increase 6, range 3.1- 9.5, SEM: 1.4) compared to the analysed naïve clones. Furthermore, validation with qPCR showed 12-fold upregulation of the GAB2gene (mean upregulation 12.7, SEM: 4.8), an adaptor protein which transmits crucial signals to activate downstream PI3K signaling with links to leukemogenesis. Conclusion Our results demonstrate that common mechanisms of resistance to BCR inhibition include both the up-regulation of B-cell receptor signaling activity and the up-regulation of anti-apoptotic molecules, particularly BCL2. More potent and more target-specific BTK inhibitors may overcome the hyperactivity of the BCR pathway. In contrast, inhibition of BCL2 with venetoclax may be beneficial in overcoming the emergent resistance to ibrutinib as these cells become more sensitive to BCL2 inhibition. These initial results support the combination of ibrutinib with the pro-apoptotic BCL2 inhibitor, venetoclax, which we are currently studying in the Bloodwise TAP CLARITY study. Disclosures Hillmen: Pharmacyclics: Research Funding; Janssen: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Research Funding.

Background Plasma cells (PCs) are heterogeneous in terms of function and lifespan. Long-lived PCs are not depleted by B-cell depletion therapy for autoimmune disorders and therefore likely to be responsible for lack of clinical response in a significant proportion of patients. Until recently [1], the phenotype of long-lived PCs was not known. Understanding the biological features of long-lived PCs could greatly facilitate identification of therapeutic targets. Long-lived PCs contribute to humoral memory, and may represent the cell of origin for myeloma while neoplastic PCs are generally distinguished by lack of CD19 expression. However, polyclonal CD19 Neg PCs constitute approximately 15–35% of total PCs in people aged over 50 years. Here we further characterize normal CD19 Neg PC populations in the context of normal and therapeutically perturbed bone marrow. Objectives To establish the phenotypic and functional differences between normal CD19 Pos and CD19 Neg human PCs Methods Extensive phenotyping of surface and intracellular proteins of normal and myeloma PCs were performed by flow cytometry. The regeneration of normal PCs was monitored following bone marrow ablation for myeloma [2] and B-cell depletion with alemtuzumab [3] in a series of clinical trials. The immunoglobulin heavy chain variable (IGHV) region of normal CD19 Pos and CD19 Neg human PCs were sequenced as described previously [4] to identify phylogenetic relationships. Results CD19 Neg PCs are phenotypically distinct from normal CD19 Pos plasma cells with weaker CD95 (p Neg PCs share some characteristics with neoplastic PCs including variable expression of CD28, CD56, and CD45, but neoplastic PCs are reliably distinguished from normal CD19 Neg PCs by differential expression of CD27, CD81 and CD117 as well as CD56 in some cases. After effective myeloablation for myeloma CD19 Pos PCs recovered in 74 out of 80 samples, while CD19 Neg PCs remained at less than 1% of total PCs in 30 out of 80 samples. Therapeutic B-cell depletion with alemtuzumab for chronic lymphocytic leukaemia results in loss of CD19 Pos PCs (p Neg PCs persist. There was little overlap between the IGHV repertoire with only 2.1 to 3.7% of unique IGHV@ rearrangements shared between the CD19 Pos and CD19 Neg fractions, suggestive of a largely discrete immunoglobulin repertoire in the two fractions at steady state. Conclusions CD19 Pos and CD19 Neg PCs are biologically distinct with different immunoglobulin repertoires and production kinetics consistent with CD19 Neg PCs having greater potential lifespan. Identifying the developmental pathway of normal CD19 Neg PC is likely to allow selectively targeting PC compartments for autoimmune disorders. References Mei HE, et al. Blood 2015, 125(11):1739–1748. Rawstron AC, et al. Journal of clinical oncology 2013, 31(20):2540–2547. Rawstron AC, et al. Blood 2004, 103(6):2027–2031. Cocco M, et al. Journal of Immunology 2012, 189(12):5773–5785. Disclosure of Interest None declared

γδ T cells commonly associate with mucosal and epithelial sites, fulfilling a variety of immunoregulatory functions. While lung γδ T cells have well-characterized pro-inflammatory activity, their potential role in the resolution of lung inflammation has yet to be explored in any detail. Indeed, given the importance of minimizing inflammation, the cellular mechanisms driving the resolution of lung inflammation are poorly understood. Using a murine model of acute Streptococcus pneumoniae-mediated lung inflammation, we now show that resolution of inflammation following bacterial clearance is associated with a > 30-fold increase in γδ T-cell number. Although inflammation eventually resolves in TCRδ−/− mice, elevated numbers of alveolar macrophages and pulmonary dendritic cells, and the appearance of well-formed granulomas in lungs of TCRδ−/− mice, together indicated a role for γδ T cells in regulating mononuclear phagocyte number. Ex vivo, both alveolar macrophages and pulmonary dendritic cells were susceptible to lung γδ T cell-mediated cytotoxicity, the first demonstration of such activity against a dendritic cell population. These findings support a model whereby expansion of γδ T cells helps restore mononuclear phagocyte numbers to homeostatic levels, protecting the lung from the consequences of inappropriate inflammation. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A polymorphism of the TNF-β gene can be detected by restriction digestion of a PCR product with NcoI. In this study we look at the risk associated with this polymorphism in a study of 69 insulin-dependent diabetes patients and 119 healthy controls. The risk was further characterized by comparison to the HLA type of the individual, since the TNF polymorphism is in linkage disequilibrium with HLA genes.

Background & Aims: Intestinal epithelial integrity and permeability is dependent on intercellular tight junction (TJ) complexes. How TJ integrity is regulated remains unclear, although phosphorylation and dephosphorylation of the integral membrane protein occludin is an important determinant of TJ formation and epithelial permeability. We have investigated the role intestinal intraepithelial lymphocytes (iIELs) play in regulating epithelial permeability in response to infection. Methods: Recombinant strains of Toxoplasma gondii were used to assess intestinal epithelial barrier function and TJ integrity in mice with intact or depleted populations of iIELs. Alterations in epithelial permeability were correlated with TJ structure and the state of phosphorylation of occludin. iIEL in vivo reconstitution experiments were used to identify the iIELs required to maintain epithelial permeability and TJ integrity. Results: In the absence of γδ + iIELs, intestinal epithelial barrier function and the ability to restrict epithelial transmigration of Toxoplasma and the unrelated intracellular bacterial pathogen Salmonella typhimurium was severely compromised. Leaky epithelium in γδ + iIEL-deficient mice was associated with the absence of phosphorylation of serine residues of occludin and lack of claudin 3 and zona occludens-1 proteins in TJ complexes. These deficiencies were attributable to the absence of a single subset of γδ T-cell receptor (TCR-Vγ7 + ) iIELs that, after reconstituting γδ iIEL-deficient mice, restored epithelial barrier function and TJ complexes, resulting in increased resistance to infection. Conclusions: These findings identify a novel role for γδ + iIELs in maintaining TJ integrity and epithelial barrier function that have implications for understanding the pathogenesis of intestinal inflammatory diseases associated with disruption of TJ complexes.

Gammadelta T cells play important but poorly defined roles in pathogen-induced immune responses and in preventing chronic inflammation and pathology. A major obstacle to defining their function is establishing the degree of functional redundancy and heterogeneity among gammadelta T cells. Using mice deficient in Vgamma1+ T cells which are a major component of the gammadelta T cell response to microbial infection, a specific immunoregulatory role for Vgamma1+ T cells in macrophage and gammadelta T cell homeostasis during infection has been established. By contrast, Vgamma1+ T cells play no significant role in pathogen containment or eradication and cannot protect mice from immune-mediated pathology. Pathogen-elicited Vgamma1+ T cells also display different functional characteristics at different stages of the host response to infection that involves unique and different populations of Vgamma1+ T cells. These findings, therefore, identify distinct and nonoverlapping roles for gammadelta T cell subsets in infection and establish the complexity and adaptability of a single population of gammadelta T cells in the host response to infection that is not predetermined, but is, instead, shaped by environmental factors.

Detailed analysis of the expression of natural killer (NK) cell activatory and inhibitory receptors by human decidual leukocyte subpopulations has not been undertaken.Expression of the natural cytotoxicity receptors NKp30, NKp44 and NKp46 by decidual leukocytes were studied by reverse transcriptase polymerase chain reaction. Expression of the killer cell Ig-like receptors CD158a and CD158b on decidual T cells were studied by flow cytometry.First trimester decidual leukocytes expressed mRNA for the NKp30 and NKp46 receptors but expression of NKp44, a marker of activated NK cells, was not detected. A mean of 11.8 and 15.8% of decidual T cells expressed CD158a or 158b, respectively, while only around 1% of peripheral blood T cells were CD158a+ or CD158b+.Like peripheral blood NK cells, decidual NK cells express the natural cytotoxicity receptors NKp30 and NKp46 but the significance of this will not become apparent until ligands for these molecules have been identified. Only a minority of decidual T cells express CD158, indicating that this is not a mechanism for inhibition of cytotoxicity mediated by all decidual T cells.

Background Eradication of minimal residual disease (MRD) is an independent predictor of survival outcome in patients with chronic lymphocytic leukaemia (CLL) receiving fludarabine and cyclophosphamide (FC) or fludarabine, cyclophosphamide, and rituximab (FCR) as first-line treatment. However, the independent prognostic relevance of MRD status in other therapeutic settings is not clear. The goal of this study was to investigate the independent importance of achieving MRD negativity in CLL on progression-free survival (PFS) and overall survival (OS) with different treatments in frontline and relapsed or refractory settings compared with known prognostic markers. Methods We included all patients at our centre in Leeds and associated hospitals in West and North Yorkshire who had completed treatment for CLL from 1996 to 2007, achieved at least a partial response, and received an MRD assessment from a bone-marrow specimen after treatment. MRD assessments were done by multiparametric flow cytometry using CD5–CD19 in combination with CD20, CD22, CD32, CD38, CD79b, and CD81 in accordance with the international harmonised approach. 133 patients (17 receiving fludarabine, 65 fludarabine-based combination therapies, 26 alemtuzumab, and 25 other treatment including chlorambucil and autologous stem-cell transplantation) were followed up for a median of 5·2 years (IQR 3·0–7·3) to assess PFS and OS. Findings MRD negativity (defined as less than one CLL cell in 10 000 leucocytes) at the end of therapy independently correlated with both PFS (hazard ratio [HR] 3·22 [95% CI 2·09–4·97], Cox proportional hazards model p vs 31) and OS (78 vs 64) than did MRD-positive individuals without such adverse cytogenetic abnormalities. Interpretation MRD status is a powerful independent predictor of survival outcome in CLL across a range of therapeutic approaches including in frontline and relapse settings. MRD negativity is the most appropriate therapeutic goal for CLL patients who are fit enough for such an approach. Funding None.

Inflammatory mediators including chemokines play a critical role in acute pancreatitis. The precise nature of early inflammatory signals within the pancreas remains, however, unclear. We examined the ability of isolated pancreatic acini to synthesize CC chemokine monocyte chemotactic protein-1 (MCP-1) and CXC chemokine cytokine-induced neutrophil chemoattractant (CINC) and the response to the secretagogue cerulein at physiological and supraphysiological concentrations. Isolated rat pancreatic acini maintained in short-term (≤48 h) primary culture constitutively synthesized MCP-1 and CINC. Cerulein (10−7M; supramaximal dose) increased production of MCP-1 but not CINC. Cerulein-induced increase in MCP-1 synthesis was accompanied by increase in nuclear factor (NF)-κB activation shown by EMSA. Pretreatment with NF-κB inhibitors N-acetylcysteine (NAC) and N-tosylphenyalanine chloromethyl ketone (TPCK) blocked cerulein-induced NF-κB activation and abolished cerulein's effect on MCP-1 synthesis. Pretreatment with calcium antagonist BAPTA-AM also blocked cerulein's effect on MCP-1 synthesis. These results indicate that isolated acini synthesize MCP-1 and CINC and support the idea of acinar-derived chemokines as early mediators of inflammatory response in acute pancreatitis. Although cerulein hyperstimulation increased MCP-1 synthesis by a calcium-dependent mechanism involving NF-κB activation, CINC synthesis was not affected. This suggests that regulation of CC and CXC chemokines within acinar cells may be quite different.

Problem: The T-cell antigen receptor (TCR) has been reported to be down-regulated on T-cells in the decidualized endometrium in early pregnancy. Method of Study: The expression of CD3 zeta a component of the TCR complex, has been investigated in human first-trimester decidual T-cells using flow cytometric analysis of permeabilized cells. Results: Levels of CD3 zeta expression were significantly lower in decidual than in peripheral T-cells from non-pregnant women, as assessed by mean fluorescence intensity (4.2 vs 5.5, logarithmic scale, P0.1). CD3 zeta was not substantially down-regulated systemically as mean cytoplasmic CD3 zeta levels did not differ significantly between peripheral blood T-cells from pregnant women and non-pregnant controls (P>0.2). CD8+ cells outnumber CD4+ cells in decidua, but neither the proportions of these two T-cell subsets positive for cytoplasmic CD3eta nor the mean levels of CD3 zeta were significantly different. Conclusions: These results indicate that human decidual T-cells do not greatly down-regulate CD3 zeta, but it is unclear if a small decrease in mean levels may be sufficient to compromise their capacity for activation.

The reverse transcription polymerase chain reaction (RT-PCR) with primers specific for each of the 14 exons of the human complement regulatory protein membrane cofactor protein (MCP;CD46) has been utilized to determine MCP mRNA transcript expression in peripheral blood mononuclear cells (PBMC). An additional transcript of a larger size than predicted was consistently detected in reactions with a sense primer for exon 7, that encodes the first alternatively spliced serine–threonine-rich region (ST-A), together with an antisense exon 12 primer. RT-PCR with primers for other exons both 5′ and 3′ of exon 7 further showed that these MCP transcripts contain additional sequences immediately both 5′ and 3′ to the exon 7-encoded sequence. Comparison of genomic DNA with cDNA by PCR, in combination with sequence analysis, demonstrated the presence of the complete invariant sequences of both introns adjacent to exon 7, i.e. intron 6 (411 bp) and intron 7 (127 bp). RT-PCR using primers specific for the intron 6 sequence, together with Southern and Northern blotting using an intron 6-specific probe, confirmed retention of this intron within a novel 4.8-kb mRNA transcript in human PBMC. Due to the presence of a stop codon within intron 6, translation would result in a novel truncated MCP isoform (MCPi) containing the four invariant short consensus repeat (SCR) regions and a unique C-terminal 39 amino acid transmembrane and cytoplasmic tail region that may promote endoplasmic reticulum retention.

Introduction Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired bone marrow disorder characterised by intravascular hemolysis and hemoglobinuria, potentially life-threatening thrombosis and an association with aplastic anemia. Most of the clinical features and complications of PNH are due to the unopposed activity of complement due to the absence of CD59 and CD55, two key regulators of complement. Eculizumab prevents the cleavage of C5 complement thereby preventing terminal complement activity and protecting PNH cells from lysis. The inhibition of C5 preserves the early part of complement pathway and leads to the build up of C3 on the PNH red cells, perhaps in part due to their lack of CD55. The majority of PNH patients receiving eculizumab have evidence of extravascular haemolysis that can be clinically significant, including with anemia, hyperbilirubinemia and in some a continued requirement for transfusions. This extravascular hemolysis in thought to be due to the C3 loading of PNH red cells. Methods We report the C3-loading of the PNH red cells from 119 patients treated with eculizumab and correlate this with hemoglobin, LDH, bilirubin, reticulocytes and transfusions. We have studied genetic polymorphisms that affect both C3 and FCγR. We have genotyped 46 eculizumab patients for a functional mutation in the C3 gene (rs2230199). The two alleles of this gene can be distinguished by the presence or absence of a HindIII restriction site that distinguishes the electophoretically slow (arg80) from the electrophoretically fast (gly80) allotype. The fast (C3F) allotype allele of this snp is associated with a range of disorders including age-related macular degeneration, IgA nephropathy, systemic vasculitis and partial lipodystrophy. APL-1 is a small cyclic peptide that binds to and inhibits the activation of complement C3. APL-2 is a large conjugate of APL-1 with enhanced bioactivity and a long systemic half-life. APL-1 and APL-2 molecules as well as other complement inhibitors were studied for lysis of red cells and C3 loading in vitro in a modified Ham test in which flow cytometry was used to identify non-lysed cells. Results Out of the 119 Eculizumab treated patients, 55 (46.2%) required at least one transfusion on treatment. 110 patients had C3 detectable by flow cytometry on their PNH red cells (mean of 19.8%; range: We studied one functional polymorphism in the Fcγ receptor but this showed no correlation with haemolytic parameters. Conversely, for the C3 polymorphism eculizumab-treated patients homozygous for the slow (C3S) allele had a significantly higher degree of C3 loading on PNH red blood cells with C3S/C3S having a mean of 33.7% C3 loaded PNH red cells (n=26), C3S/C3F 19.0% (n=19) and C3F/C3F 12.8% (n=3)(all P In vitro experiments show that inhibitors of C5, such as eculizumab, protect the PNH red cells from lysis but lead to a very rapid deposition of C3 on the PNH red cells. However both APL1 and APL2 demonstrated similar protection of PNH red cell lysis with virtually no C3 loading on PNH red cells. Conclusion A significant proportion of patients on eculizumab experience extravascular haemolysis with the majority of patients developing C3 loading. We show for the first time that a functional polymorphism in the C3 gene is associated parameters of hemolysis. The S(low) allele alters the function of C3 protein and appears to be associated with extravascular haemolysis in patients with PNH. The C3 inhibitors, APL-1 and APL-2, protect PNH red cells and prevent C3 loading in vitro and if safe to be given chronically would be expected to reduce extravascular hemolysis significantly. Disclosures: Hill: Alexion Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Kelly:Alexion: Honoraria. Richards:Alexion Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Hillmen:Alexion: Honoraria, Membership on an entity’s Board of Directors or advisory committees.