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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

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.

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.

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.

A role for NLRP3 inflammasome in recurrent and chronic inflammation was initially described in a group of rare autoinflammatory conditions, termed cryopyrin-associated periodic syndrome. Subsequently, inflammasomes have been implicated in the pathology of many common diseases, including cancer, gout and diabetes. Despite diverse pathologies, the central role of the inflammasome in innate defences and tumour elimination suggests common therapeutic approaches to reduce inflammation where appropriate.

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.

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.

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.

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.

The activity of the IgH (E mu) enhancer in the T lymphocyte lineage has been investigated using both transgenic mice and transfection studies. Thymocyte fractionation experiments indicate that a transgene consisting of the bacterial chloramphenicol acetyl transferase (CAT) gene, linked to E mu and the SV40 early promoter (E mu-CAT), is expressed only in thymocytes with a mature medullary phenotype and not in immature cells. Transfection of this same construct into two thymoma cell lines representing different stages of thymocyte development mimics the pattern of activity observed in vivo. Further transfection experiments suggest that this pattern of expression might be attributed to the differential activity of the E2E3 and octanucleotide motifs of E mu during development. In contrast, an Ig lambda transgene (linked to E mu and an Ig V lambda promoter) is expressed in the majority of thymocytes. We envisage that the different patterns of expression of the two transgenes reflect interactions between their respective promoters and the factors which are bound to E mu at different stages of thymocyte development. Although differing in their pattern of expression within the thymus, the two transgenes share the property of extinction in peripheral T lymphocytes. These results indicate that the expression of E mu-linked transgenes in the thymus cannot simply be explained by activation of the enhancer in a lymphoid progenitor cell prior to B/T lineage divergence. Rather, the enhancer (or components of it) must be independently activated (and inactivated) during T lymphocyte development. Furthermore, this activity is consistent with the developmental timing of Ig DH-JH rearrangements in these cells.

In addition to the major human immunoglobulin heavy-chain locus on chromosome 14q32.3, VH and D segments are known to be present on chromosomes 15 and 16. We have now amplified and sequenced 24 such VH segments from somatic cell hybrids and have assigned them to 15q11.2 and 16p11.2 using cosmid and yeast artificial chromosome clones. In addition, we have located a cluster of D segments on 15q11.2, previously thought to be located on 14q32.3. We propose that the segments on chromosome 16 arose by an interchromosomal duplication and identify the corresponding region on chromosome 14. Taken together with the completion of a map of the human VH locus on 14q32.3, the total number of VH segments now identified is 117. We can now account for most, if not all human germ-line VH segments.

The expression of immunoglobulin heavy-chain (IgH) genes is generally thought to be regulated by the combination of the VH promoter with the enhancer element which is located in the JH-CH intron. This is probably an oversimplification: there are cell lines that transcribe IgH genes despite the deletion of the intron-enhancer. These findings could imply that other enhancer element(s) exist in the IgH locus. Here we show that a strong B-cell-specific enhancer is indeed located at the 3'-end of the rat IgH locus, 25 kilobases downstream of C alpha. This enhancer should be retained downstream of all rearranged IgH genes, regardless of the VH or CH segment used. Taken together with analogous findings for the mouse kappa locus, the results prompt a re-evaluation of the mechanism of regulation of immunoglobulin gene transcription. Furthermore, unlike the intron-enhancer, the IgH 3' enhancer would become linked to a c-myc that rearranges into an IgH switch region. The IgH 3' enhancer could therefore play a part in the activation of the translocated c-myc genes in rat immunocytomas, mouse plasmacytomas and Burkitt lymphomas.

The IgH enhancer is a strong lymphoid-specific activator and is composed of multiple factor-binding motifs. One of these, the octamer, is common to enhancer and promoter, binds ubiquitous and lymphoid-specific factors and is able to act as a lymphoid-specific transcriptional activator. However, it is also found as an essential component of promoters active in non-lymphoid cells. From analysis of the activities of synthetic promoters, we suggest that recruitment of the lymphoid-specific octamer-binding protein next to the TATA is sufficient to create a functional lymphoid-specific promoter whereas the ubiquitous octamer binding protein is not active in single copy but can act in concert with other promoter binding factors. However, the activity of the IgH enhancer is not dependent on the octamer and we identify the E2/E3 elements as also being sufficient to confer lymphoid-specificity on a linked gene. Activity of the E2/E3 region results from the synergistic activity of the two motifs, E2 alone being able to confer a low level of activity which is dramatically increased by the adjacent E3. Thus, in the case of both the E2/E3 and the octamer motifs, interactions between adjacent elements can play a critical role in determining the tissue specificity of activity.

Adenovirus (Ad) E1A is a potent oncogene and has been shown to deregulate the expression of a large number of cellular genes leading to cellular transformation. Here we have analysed the expression of several immunomodulatory molecules on the surface of a set of human cell lines transformed with either Ad12 or Ad5. Human cells transformed with Ad12 demonstrated reduced expression of cell surface LFA-3, Fas and MHC class I when compared to Ad5-transformed cells. Furthermore, Ad12-transformed human cell lines demonstrated greater susceptibility to lysis by lymphokine-activated killer (LAK) cells, compared to Ad5-transformed human cell lines. In contrast, previous studies with rodent cells showed that both Ad5- and Ad12-transformed rat cells were susceptible to LAK cells. Thus, transformation of human cells with Ad5 or Ad12 results in differences in the expression of immunomodulatory molecules on the cell surface and differential recognition of these virus-transformed cells by immune effector cells.

The roots of cancer immunology lie in the work of some of the most influential immunologists of the last 100 years. The year 2008 sees the centenary of the birth of immunology itself, marked by the award of the Nobel Prize to Elie Metchnikoff and Paul Ehrlich in 1908 (Kaufmann, 2008). Around this time, Ehrlich put forward a hypothesis that the immune system might control tumours. The origins and progress of cancer immunology, and its associated high and low points, has been reviewed in depth, highlighting the development of ideas from Ehrlich and Medawar through to the cancer immunesurveillance hypothesis of Burnet and into the era of cellular and molecular immunology (Dunn et al., 2002). The Oncogene review articles collected here provide a glimpse at how several of the concepts of cancer immunology currently stand.

Background Deregulated expression of the transmembrane glycoprotein CDCP1 (CUB domain-containing protein-1) has been detected in several cancers including colon, lung, gastric, breast, and pancreatic carcinomas. CDCP1 has been proposed to either positively or negatively regulate tumour metastasis. In this study we assessed the role of CDCP1 in properties of cells that are directly relevant to metastasis, namely adhesion and motility. In addition, association between CDCP1 and the tetraspanin protein CD9 was investigated. Methods CDCP1 and CD9 protein expression was measured in a series of colon cancer cell lines by flow cytometry and Western blotting. Adhesion of Colo320 and SW480 cells was determined using a Matrigel adhesion assay. The chemotactic motility of SW480 cells in which CDCP1 expression had been reduced by RNA interference was analysed using the xCELLigence system Real-Time Cell Analyzer Dual Plates combined with 8 μm pore filters. Detergent-resistant membrane fractions were generated following density gradient centrifugation and the CDCP1 and CD9 protein composition of these fractions was determined by Western blotting. The potential association of the CDCP1 and CD9 proteins was assessed by co-immunoprecipitation. Results Engineered CDCP1 expression in Colo320 cells resulted in a reduction in cell adhesion to Matrigel. Treatment of SW480 cells with CDCP1 siRNA reduced serum-induced chemotaxis. CDCP1 and CD9 cell-surface protein and mRNA levels showed a positive correlation in colon cancer cell lines and the proteins formed a low-level, but detectable complex as judged by co-sedimentation of detergent lysates of HT-29 cells in sucrose gradients as well as by co-immunoprecipitation in SW480 cell lysates. Conclusions A number of recent studies have assigned a potentially important role for the cell-surface protein CDCP1 in invasion and metastasis of a several types of human cancer cells. In this study, CDCP1 was shown to modulate cell-substratum adhesion and motility in colon cancer cell lines, with some variation depending on the colon cancer cell type. CDCP1 and CD9 were co-expressed at the mRNA and protein level and we obtained evidence for the presence of a molecular complex of these proteins in SW480 colon cancer cells. Electronic supplementary material The online version of this article (doi:10.1186/1471-2407-14-754) contains supplementary material, which is available to authorized users.

Background Diversity of immunoglobulins and the T cell antigen receptors is achieved via the recombination activating gene (RAG)-mediated rearrangement of variable (V), diversity (D) and joining (J) gene segments, and this underpins the efficient recognition of a seemingly limitless array of antigens. Analysis of V(D)J recombination activity is typically performed using extrachromosomal recombination substrates that are recovered from transfected cells and selected using bacterial transformation. We have developed a two-colour fluorescence-based system that simplifies detection of both deletion and inversion joining events mediated by RAG proteins. Results This system employs two fluorescent reporter genes that differentially mark unrearranged substrates and those that have undergone RAG-mediated deletion or inversion events. The recombination products bear the hallmarks of true V(D)J recombination and activity can be detected using fluorescence microscopy or flow cytometry. Recombination events can be detected without the need for cytotoxic selection of recombination products and the system allows analysis of recombination activity using substrates integrated into the genome. Conclusions This system will be useful in the analysis and exploitation of the V(D)J recombination machinery and suggests that similar approaches could be used to replace expression of one gene with another during lymphocyte development.