Search

CD146, also known as melanoma cell adhesion molecule (MCAM), is expressed in numerous cancers and has been implicated in the regulation of metastasis. We show that CD146 negatively regulates transendothelial migration (TEM) in breast cancer. This tumour suppressor-like activity is supported by a reduction in MCAM gene expression and increased promoter methylation in tumour tissue compared to normal breast tissue. However, increased CD146/MCAM expression is associated with poor prognosis in breast cancer, a characteristic that is difficult to reconcile with inhibition of TEM by CD146 and its epigenetic silencing. Single cell transcriptome data revealed MCAM expression in multiple cell types, including the tumour vasculature and malignant epithelial cells. MCAM expressing tumour cells were in the minority and expression was associated with epithelial to mesenchymal transition (EMT). Furthermore, gene expression signatures defining invasiveness and a stem cell-like phenotype were most strongly associated with mesenchymal-like tumour cells with low levels of MCAM mRNA, likely to represent an intermediate or hybrid E/M state. Our results show that high levels of MCAM gene expression are associated with poor prognosis in breast cancer because they reflect tumour vascularisation and EMT. However, the inhibitory effects of CD146 on TEM are likely to be weakest in an intermediate state between the epithelial and mesenchymal phenotypes, consistent with highly tumourigenic nature of this population.

The immune response to melanoma improves the survival in untreated patients and predicts the response to immune checkpoint blockade. Here, we report genetic and environmental predictors of the immune response in a large primary cutaneous melanoma cohort. Bioinformatic analysis of 703 tumor transcriptomes was used to infer immune cell infiltration and to categorize tumors into immune subgroups, which were then investigated for association with biological pathways, clinicopathologic factors, and copy number alterations. Three subgroups, with “low”, “intermediate”, and “high” immune signals, were identified in primary tumors and replicated in metastatic tumors. Genes in the low subgroup were enriched for cell-cycle and metabolic pathways, whereas genes in the high subgroup were enriched for IFN and NF-κB signaling. We identified high MYC expression partially driven by amplification, HLA-B downregulation, and deletion of IFNγ and NF-κB pathway genes as the regulators of immune suppression. Furthermore, we showed that cigarette smoking, a globally detrimental environmental factor, modulates immunity, reducing the survival primarily in patients with a strong immune response. Together, these analyses identify a set of factors that can be easily assessed that may serve as predictors of response to immunotherapy in patients with melanoma.Significance:These findings identify novel genetic and environmental modulators of the immune response against primary cutaneous melanoma and predict their impact on patient survival.See related commentary by Anichini, p. 2457

Gene lists per immune cell type after successive filtration steps.

Additional information on methods used and supplementary figures and tables. Figure S1: Schematic summary of consensus clustering; Figure S2: Comparison of the newly obtained immune subgroups with two published melanoma molecular signatures; Figure S3: Whole transcriptome differences among the three immune subgroups; Figure S4: Association of GPR15 gene expression, smoking and the three immune subgroups; Table S1: Survival analysis for the immune cell scores in our data (LMC) and TCGA datasets; Table S2: Association between clinico-pathological characteristics and the three immune subgroups in the LMC; Table S3: Differences in the immune cell scores between ever and never smokers in the High Immune Subgroup; Table S4: Association between smoking and tumor histological features in the High Immune Subgroup.

List of 70 genes shared between the signature used to generate the three immune subgroups in this study and the TCGA signature as well as tables of additional results from the analysis of the 3 immune subgroups: Genes and pathways upregulated in the High and Low Immune Subgroups; List of genes significantly correlating with MYC in melanoma cell lines; Results of copy number data analysis for MYC, NF-kappa B and IFN-gamma pathways; Results of the survival analyses of environmental and clinico-pathological factors with alternative definitions of smoking variable.

The membrane-bound lymphocyte-specific protein-tyrosine kinase (Lck) triggers T cell antigen receptor signalling to initiate adaptive immune responses. Despite many structure–function studies, the mode of action of Lck and the potential role of plasma membrane lipids in regulating Lck’s activity remains elusive. Advances in molecular dynamics simulations of membrane proteins in complex lipid bilayers have opened a new perspective in gathering such information. Here, we have modelled the full-length Lck open and closed conformations using data available from different crystalographic studies and simulated its interaction with the inner leaflet of the T cell plasma membrane. In both conformations, we found that the unstructured unique domain and the structured domains including the kinase interacted with the membrane with a preference for PIP lipids. Interestingly, our simulations suggest that the Lck-SH2 domain interacts with lipids differently in the open and closed Lck conformations, demonstrating that lipid interaction can potentially regulate Lck’s conformation and in turn modulate T cell signalling. Additionally, the Lck-SH2 and kinase domain residues that significantly contacted PIP lipids are found to be conserved among the Src family of kinases, thereby potentially representing similar PIP interactions within the family.

T cell activation is dependent upon the integration of antigenic, co-stimulatory and cytokine-derived signals and the availability and acquisition of nutrients from the environment. Furthermore, T cell activation is accompanied by reprogramming of cellular metabolism to provide the energy and building blocks for proliferation, differentiation and effector function. Transforming growth factor β (TGFβ) has pleiotropic effects on T cell populations, having both an essential role in the maintenance of immune tolerance but also context-dependent pro-inflammatory functions. We set out to define the mechanisms underpinning the suppressive effects of TGFβ on mouse CD8

Microscopic ulceration is an independent predictor of melanoma death. Here, we used systems biology to query the role of host and tumour-specific processes in defining the phenotype. Albumin level as a measure of systemic inflammation was predictive of fewer tumour-infiltrating lymphocytes and poorer survival in the Leeds Melanoma Cohort. Ulcerated melanomas were thicker and more mitotically active (with corresponding transcriptomic upregulated cell cycle pathways). Sequencing identified tumoural p53 and APC mutations, and TUBB2B amplification as associated with the phenotype. Ulcerated tumours had perturbed expression of cytokine genes, consistent with protumourigenic inflammation and histological and transcriptomic evidence for reduced adaptive immune cell infiltration. Pathway/network analysis of multiomic data using neural networks highlighted a role for the β-catenin pathway in the ulceration, linking genomic changes in the tumour to immunosuppression and cell proliferation. In summary, the data suggest that ulceration is in part associated with genomic changes but that host factors also predict melanoma death with evidence of reduced immune responses to the tumour.

Summary Glioblastoma (GBM) is an aggressive cancer with a very poor prognosis. Generally viewed as weakly immunogenic, GBM responds poorly to current immunotherapies. To understand this problem more clearly we used a combination of natural killer (NK) cell functional assays together with gene and protein expression profiling to define the NK cell response to GBM and explore immunosuppression in the GBM microenvironment. In addition, we used transcriptome data from patient cohorts to classify GBM according to immunological profiles. We show that glioma stem‐like cells, a source of post‐treatment tumour recurrence, express multiple immunomodulatory cell surface molecules and are targeted in preference to normal neural progenitor cells by natural killer (NK) cells ex vivo. In contrast, GBM‐infiltrating NK cells express reduced levels of activation receptors within the tumour microenvironment, with hallmarks of transforming growth factor (TGF)‐β‐mediated inhibition. This NK cell inhibition is accompanied by expression of multiple immune checkpoint molecules on T cells. Single‐cell transcriptomics demonstrated that both tumour and haematopoietic‐derived cells in GBM express multiple, diverse mediators of immune evasion. Despite this, immunome analysis across a patient cohort identifies a spectrum of immunological activity in GBM, with active immunity marked by co‐expression of immune effector molecules and feedback inhibitory mechanisms. Our data show that GBM is recognized by the immune system but that anti‐tumour immunity is restrained by multiple immunosuppressive pathways, some of which operate in the healthy brain. The presence of immune activity in a subset of patients suggests that these patients will more probably benefit from combination immunotherapies directed against multiple immunosuppressive pathways., Our manuscript challenges the current paradigm that Glioblastoma (GBM) is a poor candidate for immunotherapy, therefore impacting on clinical approaches. We identify NK cells as a target for immunosuppression in GBM, with multiple suppressive pathways within GBM human tumours. We also identify a group of GBM patients with higher immunological activity, who will benefit from immunotherapies that target these immunosuppressive pathways.

Natural killer (NK) cells protect against intracellular infection and cancer. These properties are exploited in oncolytic virus (OV) therapy, where antiviral responses enhance anti-tumour immunity. We have analysed the mechanism by which reovirus, an oncolytic dsRNA virus, modulates human NK cell activity. Reovirus activates NK cells in a type I interferon (IFN-I) dependent manner, inducing STAT1 and STAT4 signalling in both CD56

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.

Natural killer (NK) cells protect against intracellular infection and cancer. These properties are exploited in oncolytic virus (OV) therapy, where anti-viral responses enhance anti-tumour immunity. We have analysed the mechanism by which reovirus, an oncolytic dsRNA virus, modulates human NK cell activity. Reovirus activates NK cells in a type I interferon (IFN-I) dependent manner, resulting in STAT1 and STAT4 signalling in both CD56dimand CD56brightNK cell subsets. Gene expression profiling revealed the dominance of IFN-I responses and identified induction of genes associated with NK cell cytotoxicity and cell cycle progression, with distinct responses in the CD56dimand CD56brightsubsets. However, reovirus treatment, acting via IFN-I, inhibited NK cell proliferative responses to IL-15 and was associated with reduced AKT signalling.In vivo, human CD56dimand CD56brightNK cells responded with similar kinetics to reovirus treatment, but CD56brightNK cells were transiently lost from the peripheral circulation at the peak of the IFN-I response, suggestive of their redistribution to secondary lymphoid tissue. These results show that reovirus modulates a spectrum of NK cell activityin vivo, encompassing direct action on tumour cells and the regulation of adaptive immunity. Such activity is likely to mirror NK cell responses to natural viral infection.

Summary In the Medical Research Council (MRC) Myeloma IX trial (ISRCTN684564111) patients were randomised to sodium clodronate or zoledronic acid and induction treatment: cyclophosphamide, vincristine, doxorubicin and dexamethasone (CVAD) or cyclophosphamide, thalidomide and dexamethasone (CTD) followed by autologous stem cell transplant (ASCT) in the intensive pathway; attenuated CTD or melphalan and prednisolone (MP) in the non‐intensive pathway. Subsequent randomisation allocated patients to either thalidomide or observation. The European Organisation for Research and Treatment of Cancer (EORTC) quality of life (QoL) questionnaires, QLQ‐C30 and QLQ‐MY24, were administered at baseline, 3, 6 and 12 months and annually thereafter, enabling the effect of sequential treatment on patient‐reported health‐related QoL (HR‐QoL) to be investigated. The protocol specified four subscales of interest: Pain, Fatigue, Global Health Status/Quality of Life and Physical Functioning at 3, 6 and 12 months that were compared using linear models. The intensive pathway showed significant differences in favour of CTD for Fatigue at 3 months and Physical Functioning at 12 months. The non‐intensive pathway and maintenance phase reported significant differences at 3 months; Pain (improved with attenuated CTD) and Global Health status/Quality of Life (improved with observation). The improved outcomes in MRC Myeloma IX were accompanied by some beneficial and few detrimental effects on HR‐QoL.

Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable.Rationalin silicodesign andin vitroscreens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic “swine” H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance.

The immune response to melanoma improves the survival in untreated patients and predicts the response to immune checkpoint blockade. Here, we report genetic and environmental predictors of the immune response in a large primary cutaneous melanoma cohort. Bioinformatic analysis of 703 tumor transcriptomes was used to infer immune cell infiltration and to categorize tumors into immune subgroups, which were then investigated for association with biological pathways, clinicopathologic factors, and copy number alterations. Three subgroups, with “low”, “intermediate”, and “high” immune signals, were identified in primary tumors and replicated in metastatic tumors. Genes in the low subgroup were enriched for cell-cycle and metabolic pathways, whereas genes in the high subgroup were enriched for IFN and NF-κB signaling. We identified high MYC expression partially driven by amplification, HLA-B downregulation, and deletion of IFNγ and NF-κB pathway genes as the regulators of immune suppression. Furthermore, we showed that cigarette smoking, a globally detrimental environmental factor, modulates immunity, reducing the survival primarily in patients with a strong immune response. Together, these analyses identify a set of factors that can be easily assessed that may serve as predictors of response to immunotherapy in patients with melanoma. Significance: These findings identify novel genetic and environmental modulators of the immune response against primary cutaneous melanoma and predict their impact on patient survival. See related commentary by Anichini, p. 2457

Following the publication of this article the authors noted that the MRD data under the Table 1 column “Remark” of Aspire should go to that of Pollux. The authors wish to apologize for any inconvenience caused. The corrected table is attached to this correction.

Summary Glycogen synthase kinase-3 (GSK-3) is a positive regulator of PD-1 expression in CD8+ T cells and GSK-3 inhibition enhances T cell function and is effective in the control of tumor growth. GSK-3 has two co-expressed isoforms, GSK-3α and GSK-3β. Using conditional gene targeting, we demonstrate that both isoforms contribute to T cell function to different degrees. Gsk3b−/− mice suppressed tumor growth to the same degree as Gsk3a/b−/− mice, whereas Gsk3a−/− mice behaved similarly to wild-type, revealing an important role for GSK-3β in regulating T cell-mediated anti-tumor immunity. The individual GSK-3α and β isoforms have differential effects on PD-1, IFNγ, and granzyme B expression and operate in synergy to control PD-1 expression and the infiltration of tumors with CD4 and CD8 T cells. Our data reveal a complex interplay of the GSK-3 isoforms in the control of tumor immunity and highlight non-redundant activity of GSK-3 isoforms in T cells, with implications for immunotherapy., Graphical abstract, Highlights • Conditional knockouts reveal a non-redundant role for GSK-3α and GSK-3β in T cells. • GSK-3α and GSK-3β act together to enhance PD-1 expression. • GSK-3β depletion alone enhances T cell mediated tumor rejection. • GSK-3β plays an important role in regulating T cell-mediated anti-tumor immunity., Cancer; Cell biology; Functional aspects of cell biology; Immunology

Summary Human natural killer (NK) cells play an important role in anti-viral immunity. However, studying their activation kinetics during infection is highly problematic. A clinical trial of a therapeutic virus provided an opportunity to study human NK cell activation in vivo in a controlled manner. Ten colorectal cancer patients with liver metastases received between one and five doses of oncolytic reovirus prior to surgical resection of their tumour. NK cell surface expression of the interferon-inducible molecules CD69 and tetherin peaked 24–48 h post-infection, coincident with a peak of interferon-induced gene expression. The interferon response and NK cell activation were transient, declining by 96 h post-infection. Furthermore, neither NK cell activation nor the interferon response were sustained in patients undergoing multiple rounds of virus treatment. These results show that reovirus modulates human NK cell activity in vivo and suggest that this may contribute to any therapeutic effect of this oncolytic virus. Detection of a single, transient peak of activation, despite multiple treatment rounds, has implications for the design of reovirus-based therapy. Furthermore, our results suggest the existence of a post-infection refractory period when the interferon response and NK cell activation are blunted. This refractory period has been observed previously in animal models and may underlie the enhanced susceptibility to secondary infections that is seen following viral infection.

BACKGROUND: The NLRP3-inflammasome, implicated in the pathogenesis of several inflammatory disorders, has been analysed in rheumatoid arthritis (RA). METHODS: Relative gene expression of NLRP3-inflammasome components was characterised in PBMCs of 29 patients receiving infliximab. A total of 1278 Caucasian patients with RA from the Biologics in Rheumatoid Arthritis Genetics and Genomics Study Syndicate (BRAGGSS) cohort receiving tumour necrosis factor (TNF) antagonists (infliximab, adalimumab and etanercept) were genotyped for 34 single nucleotide polymorphisms (SNPs), spanning the genes NLRP3, MEFV and CARD8. Regression analyses were performed to test for association between genotype and susceptibility and treatment response (disease activity score across 28 joints (DAS28) and EULAR improvement criteria) at 6 months, with secondary expression quantitative trait loci (eQTL) analyses. RESULTS: At baseline, gene expression of ASC, MEFV, NLRP3-FL, NLRP3-SL and CASP1 were significantly higher compared with controls whereas CARD8 was lower in the patients. Caspase-1 and interleukin-18 levels were significantly raised in patients with RA. SNPs in NLRP3 showed association with RA susceptibility and EULAR response to anti-TNF in the BRAGGSS cohort, and in monocytes but not B cells, in eQTL analysis of 283 healthy controls. CARD8 SNPs were associated with RA susceptibility and DAS28 improvement in response to anti-TNF and eQTL effects in monocytes and B cells. CONCLUSIONS: This study found evidence of modulation of the NLRP3-inflammasome in patients with RA prior to receiving infliximab and some evidence of association for SNPs at NLRP3 and CARD8 loci with RA susceptibility and response to anti-TNF. The SNPs associated with susceptibility/response are not the main eQTL variants for either locus, and the associations with treatment response require replication in an independent cohort.

Purpose: Epigenetic dysregulation is known to be an important contributor to myeloma pathogenesis but, unlike other B-cell malignancies, the full spectrum of somatic mutations in epigenetic modifiers has not been reported previously. We sought to address this using the results from whole-exome sequencing in the context of a large prospective clinical trial of newly diagnosed patients and targeted sequencing in a cohort of previously treated patients for comparison. Experimental Design: Whole-exome sequencing analysis of 463 presenting myeloma cases entered in the UK NCRI Myeloma XI study and targeted sequencing analysis of 156 previously treated cases from the University of Arkansas for Medical Sciences (Little Rock, AR). We correlated the presence of mutations with clinical outcome from diagnosis and compared the mutations found at diagnosis with later stages of disease. Results: In diagnostic myeloma patient samples, we identify significant mutations in genes encoding the histone 1 linker protein, previously identified in other B-cell malignancies. Our data suggest an adverse prognostic impact from the presence of lesions in genes encoding DNA methylation modifiers and the histone demethylase KDM6A/UTX. The frequency of mutations in epigenetic modifiers appears to increase following treatment most notably in genes encoding histone methyltransferases and DNA methylation modifiers. Conclusions: Numerous mutations identified raise the possibility of targeted treatment strategies for patients either at diagnosis or relapse supporting the use of sequencing-based diagnostics in myeloma to help guide therapy as more epigenetic targeted agents become available. Clin Cancer Res; 22(23); 5783–94. ©2016 AACR.

Mutations in the RAS family of oncogenes are highly prevalent in human cancer and, amongst its manifold effects, oncogenic RAS impairs the expression of components of the antigen presentation pathway. This allows evasion of cytotoxic T lymphocytes (CTL). CTL and natural killer (NK) cells are reciprocally regulated by MHC class I molecules and any gain in CTL recognition obtained by therapeutic inactivation of oncogenic RAS may be offset by reduced NK cell activation. We have investigated the consequences of targeted inactivation of oncogenic RAS on the recognition by both CTL and NK cells. Inactivation of oncogenic RAS, either by genetic deletion or inactivation with an inducible intracellular domain antibody (iDAb), increased MHC class I expression in human colorectal cell lines. The common RAS mutations, at codons 12, 13 and 61, all inhibited antigen presentation. Although MHC class I modulates the activity of both CTL and NK cells, the enhanced MHC class I expression resulting from inactivation of mutant KRAS did not significantly affect the in vitro recognition of these cell lines by either class of cytotoxic lymphocyte. These results show that oncogenic RAS and its downstream signalling pathways modulate the antigen presentation pathway and that this inhibition is reversible. However, the magnitude of these effects was not sufficient to alter the in vitro recognition of tumour cell lines by either CTL or NK cells.

NK cell activation is negatively regulated by the expression of target cell MHC class I molecules. We show that this relationship is nonlinear due to an NK cell activation/inhibition threshold. Ewing’s sarcoma family tumor cell monolayers, which were highly susceptible to NK cells in vitro, developed a highly resistant phenotype when cultured as three-dimensional multicellular tumor spheroid structures. This suggested that tumor architecture is likely to influence the susceptibility to NK cells in vivo. Resistance of the multicellular tumor spheroid was associated with the increased expression of MHC class I molecules and greatly reduced NK cell activation, implying that a threshold of NK cell activation/inhibition had been crossed. Reducing MHC class I expression on Ewing’s sarcoma family tumor monolayers did not alter their susceptibility to NK cells, whereas increased expression of MHC class I rendered them resistant and allowed the threshold point to be identified. This threshold, as defined by MHC class I expression, was predictive of the number of NK-resistant target cells within a population. A threshold permits modest changes in the target cell surface phenotype to profoundly alter the susceptibility to NK cells. Whereas this allows for the efficient detection of target cells, it also provides a route for pathogens and tumors to evade NK cell attack.

Allogeneic haematopoietic cell transplantation is an established curative treatment modality for patients with malignant and non-malignant haematological disorders. Since the first related umbilical cord blood transplant (UCBT) in 1988, the use of UCB as a stem cell source for transplantation has become a standard practice in many countries, with approximately 8000 such transplants having been performed worldwide to date.

AlloSCT is a potentially curative procedure for haematological malignancies and marrow failure syndromes. However, unlike leukaemia and lymphoproliferative disorders, AlloSCT has yet to find its place in the clinical management of patients with multiple myeloma. AlloSCT in multiple myeloma is associated with a high procedure-related mortality (TRM up to 35%) when full-intensity conditioning is used and only up to 36% of cases show long-term disease-free survival. The introduction of reduced intensity conditioning AlloSCT, more recently following an autologous SCT, has reduced the TRM to

Natural killer (NK) cells are lymphocytes with an innate ability to recognize and kill infected cells and tumour cells. Unlike B and T cells, NK cells do not express an antigen receptor. Instead, NK cells detect changes in the phenotype of the target cell surface; malignant transformation or infection resulting in the loss or gain of particular molecules that are detected by inhibitory or activating receptors on the NK cell surface. The identification and characterization of NK cells and their receptors was made possible by monoclonal antibody technology. The ease with which genes and gene products can now be identified and manipulated has accelerated our understanding of NK cell function. Furthermore, gene and protein profiling studies are beginning to refine our understanding of NK cells, their interactions with other cells and their effector mechanisms. This review illustrates some of the basic features of NK cell biology and highlights the contribution made by post-genomic technology in defining the molecular mechanisms by which NK cells identify and kill susceptible targets.

Peripheral T-cell lymphomas (PTCL) are a rare and heterogeneous subset of lymphomas with a poorer prognosis compared with B-cell lymphomas. We conducted a retrospective study of 82 patients who received high-dose therapy for PTCL (autologous SCT (ASCT) N=64; allogeneic SCT (Allo-SCT) N=18). With a median follow-up from ASCT of 37 months from transplant, 33 patients were alive; 20 died of progressive disease, 10 died from non-relapse mortality (NRM) with 1 unknown cause. Three-year overall survival (OS) and progression-free survival (PFS) were 53% (95% confidence interval (CI) 42, 67) and 50% (95% CI 39, 64), respectively. Factors significantly affecting OS and PFS on univariate analysis were histological subtype and chemotherapy sensitivity. In a multivariate analysis, the only factor with significant impact was chemotherapy sensitivity. After a median follow-up from Allo-SCT of 57 months, five patients were alive; five died of progressive disease and eight died from NRM. The 3-year OS and PFS were 39% (95% CI 22, 69) and 33% (95% CI 17, 64), respectively, and the 3-year relapse rate was 28% (95% CI 6, 50). These results demonstrate that high-dose chemotherapy with autologous stem cell rescue has a substantial role in the management of T-cell lymphoma. The use of full-intensity allogeneic transplantation is limited by high transplant-related mortality, and exploration of reduced intensity regimens is warranted.

To the editor: Treatment of the glycosylphosphatidylinositol (GPI) anchor deficiency paroxysmal nocturnal hemoglobinuria (PNH) has been revolutionized by the use of the anti-C5 antibody eculizumab, which blocks complement-mediated hemolysis and the associated pathology.[1][1],[2][2] In addition to

Analysis of outcome following allogeneic haemopoietic stem cell transplantation for myeloma using myeloablative conditioning - evidence for a superior outcome using melphalan combined with total body irradiation We have undertaken a retrospective multicentre analysis of 139 patients (median age 44.4 years) undergoing allogeneic haematopoietic stem cell transplantation (HSCT) for multiple myeloma using myeloablative conditioning. The majority of patients received total body irradiation (TBI) combined with either melphalan (56.9%) or cyclophosphamide (28.5%). Overall, transplant-related mortality (TRM) was 37.9% at 1 year and was not significantly different for patients receiving melphalan/TBI compared with cyclophosphamide/TBI. The overall complete remission (CR) rate, including patients in CR at the time of transplant, was greater for patients receiving melphalan/TBI (64.7%) compared with cyclophosphamide/TBI (47.2%)(P = 0.085). A significantly higher proportion of patients with continuing disease at the time of transplant achieved CR post-transplant following melphalan/TBI conditioning compared with cyclophosphamide/TBI (52.9% and 33.4% respectively, P = 0.009). Relapse/progression rates at 5 years were significantly lower for melphalan/TBI (36.7%) compared with cyclophosphamide/TBI (80.8%, P < 0.0001) and remained significant in multivariate analysis. This resulted in an overall survival at 5 years of 44.1% and 28.1% for melphalan/TBI and cyclophosphamide/TBI, respectively (P = 0.059). These results demonstrate that the type of conditioning for sibling allogeneic HSCT for myeloma has a major effect on transplant outcome.

Interactions between natural killer (NK) cells and dendritic cells (DCs) aid DC maturation and promote T-cell responses. Here, we have analyzed the response of human NK cells to tumor cells, and we identify a pathway by which NK-DC interactions occur. Gene expression profiling of tumor-responsive NK cells identified the very rapid induction of TNF superfamily member 14 [TNFSF14; also known as homologous to lymphotoxins, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes (LIGHT)], a cytokine implicated in the enhancement of antitumor responses. TNFSF14 protein expression was induced by three primary mechanisms of NK cell activation, namely, via the engagement of CD16, by the synergistic activity of multiple target cell-sensing NK-cell activation receptors, and by the cytokines IL-2 and IL-15. For antitumor responses, TNFSF14 was preferentially produced by the licensed NK-cell population, defined by the expression of inhibitory receptors specific for self-MHC class I molecules. In contrast, IL-2 and IL-15 treatment induced TNFSF14 production by both licensed and unlicensed NK cells, reflecting the ability of proinflammatory conditions to override the licensing mechanism. Importantly, both tumor- and cytokine-activated NK cells induced DC maturation in a TNFSF14-dependent manner. The coupling of TNFSF14 production to tumor-sensing NK-cell activation receptors links the tumor immune surveillance function of NK cells to DC maturation and adaptive immunity. Furthermore, regulation by NK cell licensing helps to safeguard against TNFSF14 production in response to healthy tissues.

The use of reduced intensity conditioning (RIC) with alemtuzumab is associated with a higher rate of cytomegalovirus (CMV) reactivation than RIC without alemtuzumab,1 especially when combined with fludarabine.2 Alemtuzumab, used as in vivo T-cell depletion, has a long plasma half-life, resulting in prolonged lymphopaenia. In 16 patients receiving allogeneic stem cell transplantation (allo-SCT) with in vivo alemtuzumab, the half-life was estimated at 15–21 days with a median total lymphocyte count (TLC) at 6 months of only 0.5 × 109/l.3 Morris et al.4 reported lympholytic concentrations of alemtuzumab for 56 days after RIC in patients who were treated with 100 mg in vivo alemtuzumab (n=10).4 A sustained recovery of median TLC to >1 × 109/l was reached by 4 months in the myeloablative conditioning (MAC) group but not until 9 months in the RIC group. The higher rate of infective complications (for example, CMV and adenovirus) reported in T-cell-depleted RIC has, therefore, been attributed to delayed immune reconstitution.

The natural killer cell gene complex on human chromosome 12p12-13 encodes several C-type lectin receptor genes expressed by NK cells and other hematopoietic cells. We have identified a novel receptor gene in this region encoding a putative type II transmembrane glycoprotein. The product is 54% identical to the rat mast cell function-associated antigen (MAFA), which inhibits mast cell activation by IgE. The human MAFA-like receptor (MAFA-L) and the rat MAFA protein are expressed by basophils and both have an immunoreceptor tyrosine-based inhibitory motif in the cytoplasmic tail, consistent with an inhibitory role in basophil activation. Unlike rat MAFA, expression of the MAFA-L gene is not limited to mast cells and basophils. In common with other genes in the NK cell gene complex MAFA-L is also expressed by natural killer cells as well as the monocyte-like cell-line U937. Expression in NK cells is restricted to peripheral blood NK cells, decidual NK cells do not express MAFA-L. While MAFA-L and rat MAFA might have a similar role in basophils, the expression of MAFA-L in other cell types implies additional functions for this molecule. The presence of the MAFA-L gene in the human NK cell complex indicates that this locus encodes C-type lectin receptors expressed by a variety of cells important in host defense.

In paroxysmal nocturnal hemoglobinuria (PNH), hematopoietic cells lacking glycosylphosphatidylinositol (GPI)-linked proteins on their surface (GPI(neg)) exist alongside normal (GPI+) cells. Analysis of natural killer (NK) cell subsets in 47 PNH patients revealed that the ratio of CD56(bright):CD56(dim) NK cells differed in the GPI+ and GPI(neg) populations, with GPI(neg)CD56(bright) NK cells significantly more abundant in peripheral blood than their normal GPI+ counterparts. Indeed, GPI+CD56(bright) NK cells were not detected in the peripheral blood of some patients, suggesting their trafficking to a niche unavailable to the GPI(neg)CD56(bright) NK cell population. Defective cellular trafficking in this disease was supported by findings showing differential chemokine receptor expression between GPI+ and GPI(neg) NK cells and impaired stromal cell-derived factor 1 (SDF-1)-induced chemotaxis of GPI(neg) NK cells. Our results indicate a role for GPI-linked proteins in NK cell subset homeostasis and suggest that differential chemokine responses might contribute to the balance of GPI+ and GPI(neg) populations in this disease.

EBV-associated post-transplant lymphoproliferative disease (PTLD) following Alemtuzumab-based allo-SCT is a relatively uncommon and challenging clinical problem but has not received detailed study in a large cohort. Quantitative-PCR (qPCR) monitoring for EBV reactivation post allo-SCT is now commonplace but its diagnostic and predictive value remains unclear. Sixty-nine patients with PTLD following Alemtuzumab-based allo-SCT were studied. Marked clinicopathological heterogeneity was evident; lymphadenopathy was frequently absent, whereas advanced extranodal disease was common. The median viral load at clinical presentation was 49 300 copies/mL (50-65 200 000 copies/mL) and, notably, 23% and 45% of cases, respectively, had 10 000 and 40 000 copies/mL. The overall response rate to rituximab as first-line therapy was 70%. For rituximab failures, chemotherapy was ineffectual but DLIs were successful. A four-parameter prognostic index predicted response to therapy (OR 0.30 (0.12-0.74); P=0.009] and PTLD mortality (hazard ratio (HR) 1.81 (1.12-2.93) P=0.02) on multivariate analysis. This is the largest detailed series of EBV-associated PTLD after allo-SCT. At clinical presentation, EBV-qPCR values are frequently below customary thresholds for pre-emptive therapy, challenging current paradigms for monitoring and intervention. A four-point score identifies a proportion of patients at risk of rituximab-refractory disease for whom alternative therapy is needed.

This retrospective national study compared the use of alemtuzumab-based conditioning regimens for hematopoietic SCT (HSCT) in acquired severe aplastic anemia with antithymocyte globulin (ATG)-based regimens. One hundred patients received alemtuzumab and 55 ATG-based regimens. A matched sibling donor (MSD) was used in 87 (56%), matched unrelated donor (MUD) in 60 (39%) and other related or mismatched unrelated donor (UD) in 8 (5%) patients. Engraftment failure occurred in 9% of the alemtuzumab group and 11% of the ATG group. Five-year OS was 90% for the alemtuzumab and 79% for the ATG groups, P=0.11. For UD HSCT, OS of patients was better when using alemtuzumab (88%) compared with ATG (57%), P=0.026, although smaller numbers of patients received ATG. Similar outcomes for MSD HSCT using alemtuzumab or ATG were seen (91% vs 85%, respectively, P=0.562). A lower risk of chronic GVHD (cGVHD) was observed in the alemtuzumab group (11% vs 26%, P=0.031). On multivariate analysis, use of BM as stem cell source was associated with better OS and EFS, and less acute and cGVHD; young age was associated with better EFS and lower risk of graft failure. This large study confirms successful avoidance of irradiation in the conditioning regimens for MUD HSCT patients.

Mice carrying transgenic human immunoglobulin gene miniloci can be used for the production of human monoclonal antibodies. The human variable region (V) gene segments in these miniloci undergo productive rearrangement in mouse lymphoid tissue to yield a population of B lymphocytes expressing a repertoire of antibodies. Many of the miniloci studied to date have included only a small number of germline gene segments in an artificially compact configuration. Here we describe the use of the bacteriophage P1 cloning system to create mice carrying the core region of the human immunoglobulin heavy chain (IgH) locus. Three P1 clones carrying overlapping regions of the human IgH locus (spanning the five J{sub H}-proximal V{sub H} segments, the entire D{sub H} and J{sub H} clusters, and the C{mu} and C{delta} constant regions) were injected into mouse eggs and appear to have reconstituted the core region of the locus (>180 kb) following homologous recombination with each other. While this translocus yielded a titer of serum immunoglobulin similar to that obtained with a smaller plasmid-basid minilocus, the P1-based locus gave rise to substantially greater diversification by somatic hypermutation. Such diversification is important for obtaining high-affinity antibodies. The results show the usefulness of the P1 system inmore » facilitating the manipulation and recreation of large transgenes. 37 refs., 7 figs., 1 tab.« less

Reovirus is a promising oncolytic virus, acting by both direct and immune-mediated mechanisms, although its potential may be limited by inactivation after systemic delivery. Our study addressed whether systemically delivered reovirus might be shielded from neutralising antibodies by cell carriage and whether virus-loaded blood or hepatic innate immune effector cells become activated to kill colorectal cancer cells metastatic to the liver in human systems. We found that reovirus was directly cytotoxic against tumour cells but not against fresh hepatocytes. Although direct tumour cell killing by neat virus was significantly reduced in the presence of neutralising serum, reovirus was protected when loaded onto peripheral blood mononuclear cells, which may carry virus after intravenous administration in patients. As well as handing off virus for direct oncolytic killing, natural killer (NK) cells within reovirus-treated blood mononuclear cells were stimulated to kill tumour targets, but not normal hepatocytes, in a Type I interferon-dependent manner. Similarly, NK cells within liver mononuclear cells became selectively cytotoxic towards tumour cells when activated by reovirus. Hence, intravenous reovirus may evade neutralisation by serum via binding to circulating mononuclear cells, and this blood cell carriage has the potential to investigate both direct and innate immune-mediated therapy against human colorectal or other cancers metastatic to the liver.

Myeloablative allo-SCT decreases relapse incidence (RI) in ALL. Reduced intensity conditioning (RIC) may extend allo-SCT to older and less fit patients. Sixty-nine ALL patients reported to the BSBMT underwent fludarabine-based RIC allo-SCT, 38 from unrelated donors (UD). Forty-four patients received alemtuzumab. ALL was in CR in 64 patients (93%). This was a second or third SCT in 23 patients. Two-year OS and PFS were 36% and 32%, respectively. In multivariate analysis male recipients demonstrated better OS and PFS (hazard ratio (HR) = 0.42, P = 0.008 and HR = 0.45, P = 0.012, respectively). Two-year TRM was 29%: higher with younger age (HR = 0.97/year, P = 0.041), female recipient (HR = 2.55, P = 0.049) and increasing grade of acute GVHD (HR = 1.87, P = 0.001). Two-year RI was 38% and was lower in patients with acute and chronic GVHD (HR = 0.62 per increasing grade, P = 0.035 and HR = 0.52, P = 0.025, respectively). Long-term ALL-free survival is achievable following fludarabine-based RIC allo-SCT. The association between GVHD and decreased RI suggests the presence of a GVL effect.

We describe 20 patients with myeloma and 1 with primary amyloidosis from 15 centres, all with advanced renal failure, most of whom had PBSC mobilised using plerixafor following previous failed mobilisation by conventional means (plerixafor used up-front for 4 patients). For 15 patients, the plerixafor dose was reduced to 0.16 mg/kg/day, with a subsequent dose increase in one case to 0.24 mg/kg/day. The remaining six patients received a standard plerixafor dosage at 0.24 mg/kg/day. Scheduling of plerixafor and apheresis around dialysis was generally straightforward. Following plerixafor administration, all patients underwent apheresis. A median CD34+ cell dose of 4.6 x 10(6) per kg was achieved after 1 (n=7), 2 (n=10), 3 (n=3) or 4 (n=1) aphereses. Only one patient failed to achieve a sufficient cell dose for transplant: she subsequently underwent delayed re-mobilisation using G-CSF with plerixafor 0.24 mg/kg/day, resulting in a CD34+ cell dose of 2.12 x 10(6)/kg. Sixteen patients experienced no plerixafor toxicities; five had mild-to-moderate gastrointestinal symptoms that did not prevent apheresis. Fifteen patients have progressed to autologous transplant, of whom 12 remain alive without disease progression. Two patients recovered endogenous renal function post autograft, and a third underwent successful renal transplantation. Plerixafor is highly effective in mobilising PBSC in this difficult patient group. Bone Marrow Transplantation (2012) 47, 18-23; doi: 10.1038/bmt.2011.9; published online 28 February 2011

Immune evasion is now recognized as a key feature of cancer progression. In animal models, the activity of cytotoxic lymphocytes is suppressed in the tumour microenvironment by the immunosuppressive cytokine, Transforming Growth Factor (TGF)-β. Release from TGF-β-mediated inhibition restores anti-tumour immunity, suggesting a therapeutic strategy for human cancer. We demonstrate that human natural killer (NK) cells are inhibited in a TGF-β dependent manner following chronic contact-dependent interactions with tumour cells in vitro. In vivo, NK cell inhibition was localised to the human tumour microenvironment and primary ovarian tumours conferred TGF-β dependent inhibition upon autologous NK cells ex vivo. TGF-β antagonized the interleukin (IL)-15 induced proliferation and gene expression associated with NK cell activation, inhibiting the expression of both NK cell activation receptor molecules and components of the cytotoxic apparatus. Interleukin-15 also promotes NK cell survival and IL-15 excluded the pro-apoptotic transcription factor FOXO3 from the nucleus. However, this IL-15 mediated pathway was unaffected by TGF-β treatment, allowing NK cell survival. This suggested that NK cells in the tumour microenvironment might have their activity restored by TGF-β blockade and both anti-TGF-β antibodies and a small molecule inhibitor of TGF-β signalling restored the effector function of NK cells inhibited by autologous tumour cells. Thus, TGF-β blunts NK cell activation within the human tumour microenvironment but this evasion mechanism can be therapeutically targeted, boosting anti-tumour immunity.

The clinical course of 2009 H1N1 influenza in Allo-SCT patients is unknown. Data were collected in the UK from October 2009 to April 2010 on laboratory-confirmed cases of H1N1 influenza in Allo-SCT recipients. H1N1 infection was diagnosed in 60 patients, median age 42 years, at a median of 10 months post-SCT. Twenty-one patients (35%) developed pneumonia and nine (15%) required admission to intensive care units. Actuarial mortality was 7% at 28 days and 19% 4 months post-diagnosis of 2009 H1N1 influenza. Increasing age and pre-existing lung disease were risk factors for pneumonia (P=0.006 and 0.037, respectively); older age was a risk factor for death (P=0.012). Morbidity and mortality from 2009 H1N1 influenza in SCT patients exceeds that of immunocompetent patients, but parallels that in other critically ill hospitalised cohorts; the elderly and those with chronic pulmonary disease are at greatest risk.