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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

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.

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

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. Rational in silico design and in vitro screens 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., Author summary "Swine flu" illustrated that the spread of influenza pandemics in the modern era is rapid, making antiviral drugs the best way of limiting disease. One proven influenza drug target is the M2 proton channel, which plays an essential role during virus entry. However, resistance against licensed drugs targeting this protein is now ubiquitous, largely due to an S31N change in the M2 sequence. Understandably, considerable effort has focused on developing M2-N31 inhibitors, yet this has been hampered by controversy surrounding two potential drug binding sites. Here, we show that both sites can in fact be targeted by new M2-N31 inhibitors, generating synergistic antiviral effects. Developing such drug combinations should improve patient outcomes and minimise the emergence of future drug resistance.

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.

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.

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.

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

A complete map of the human immunoglobulin VH locus on chromosome 14 has recently been constructed. The locus is 1100kb in length and contains 51 functional VH segments interspersed amongst a similar number of pseudogenes. Here, Graham Cook and Ian Tomlinson review the organization of the locus, its polymorphism and the repertoire it encodes.

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

Analysis of the telomeric region of chromosome 14q has enabled us to complete a map of the immunoglobulin VH locus which accounts for almost all VH segments known to rearrange in B-lymphocytes. The human germline VH repertoire consists of approximately 50 functional VH segments--the exact number depending on the haplotype--spanning 1,100 kilobases upstream of the JH segments. A yeast artificial chromosome used to map these segments was isolated by its ability to provide telomere activity in yeast, suggesting that the VH locus may be located within a few kilobases of the 14q telomere. The limited structural diversity encoded by the functional VH segments demonstrates the importance of combinatorial diversity produced by VDJ joining and the association of heavy and light chains in producing the human antibody repertoire.

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.

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.

Purpose: This phase I study in patients with metastatic melanoma (MM) and renal cell carcinoma (RCC) evaluated the safety and maximum tolerated dose (MTD), pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of s.c. treatment of human recombinant interleukin 21 (IL-21). Experimental Design: Phase I dose-escalation trial with treatment of three to six patients at each dose level, escalating from 3 to 300 μg/kg. Treatment was administered s.c. on an outpatient basis 3 days per week for 8 or 16 weeks. Results: Twenty-six patients entered the study. Recombinant IL-21 was generally well tolerated, and dose-limiting toxicities (DLT) were first seen at dose levels of 200 and 300 μg/kg. The following four DLTs were observed in three patients: increased transaminases, increased hyperbilirubinemia, hypersensitivity reaction, and lethargy. The MTD was declared to be 200 μg/kg, although five of seven patients at the 300 μg/kg dose level experienced no DLTs. A treatment-related effect on soluble CD25 was observed at all dose levels and increased with dose level. Furthermore, higher doses induced interferon-γ, perforin, and granzyme B mRNA expression in peripheral blood, and granzyme B protein expression in both CD8+ T cells and natural killer cells, consistent with the activation of cytotoxic lymphocytes. Three patients, one patient with MM and two with RCC, obtained a partial response. Conclusion: Outpatient treatment with s.c. administered IL-21 was tolerated and had dose-dependent pharmacodynamics. rIL-21 showed antitumor activity in patients with MM and RCC. Clin Cancer Res; 16(21); 5312–9. ©2010 AACR.

Prognostic impact of serum ferritin concentration on survival following reduced-intensity conditioned allogeneic haemopoietic SCT

Fifty-one patients with primary myelofibrosis (PMF) received allogeneic haematopoietic stem cell transplants from related (n=33) or unrelated (n=18) donors. Twenty-seven patients, 19-54 years old, were prepared with myeloablative regimens including CY plus BU (n=4) or TBI (n=23). Twenty-four patients, 40-64 years old, received reduced-intensity conditioning (RIC) regimens. All RIC regimens contained fludarabine, combined with melphalan (n=19) or BU (n=5), and alemtuzumab or anti-thymocyte globulin (ATG) in the majority (n=19). Four patients (17%) in the RIC group had primary graft failure. Previous splenectomy reduced time to engraftment in the RIC group (13 versus 20 days; P=0.008). For MA and RIC groups, respectively, at 3 years, overall survival rates were 44 and 31% (P=0.67), progression-free survival 44 and 24% (P=0.87), and actuarial relapse rates 15 and 46% (P=0.06). Non-relapse mortality at 3 years was 41% for the myeloablative and 32% for the RIC group. Acute GVHD occurred in 29 and 38% of patients in the myeloablative and RIC groups, respectively. Extensive chronic GVHD developed in 30 and 35% of evaluable patients, respectively.

NK cells induce apoptosis in target cells via the perforin-mediated delivery of granzyme molecules. Cytotoxic human NK cells can be generated by IL-15-mediated differentiation of CD34+ cells in vitro and these cultures have been used extensively to analyze the development of the NK cell surface phenotype. We have used NK cell differentiation in vitro together with protease-deficient human NK cells to analyze the acquisition of the cytotoxic phenotype. Granzymes are synthesized as inactive zymogens and are proteolytically activated by the cysteine protease cathepsin C. Cathepsin C is also synthesized as a zymogen and activated by proteolysis. We show that human NK cells generated in vitro undergo granule exocytosis and induce the caspase cascade in target cells. IL-15 and stem cell factor (IL-15 plus SCF) induced the expression of the granzyme B and perforin genes and the activation of cathepsin C and granzyme B zymogens. Perforin activation is also mediated by a cysteine protease and IL-15 plus SCF-mediated differentiation was accompanied by perforin processing. However, cathepsin C-deficient human NK cells revealed that perforin processing could occur in the absence of cathepsin C activity. The combination of IL-15 plus SCF is therefore sufficient to coordinate the development of the NK cell surface phenotype with the expression and proteolytic activation of the cytotoxic machinery, reflecting the central role of IL-15 in NK cell development.

ATL-104 is a potent mitogen for epithelial cells of the gastrointestinal tract. In animal models, ATL-104 aids regeneration of the gastrointestinal tract after treatment with chemotherapeutic agents. The effect of ATL-104 on mucositis in patients requiring high-dose melphalan or BEAM before peripheral blood SCT (PBSCT) was investigated in a randomized, placebo-controlled, double-blind, two-part study. Patients were randomized to ATL-104 (50, 100 or 150 mg) or placebo once daily for 3 days before chemotherapy and 3 days after PBSCT. Part one of the study was a dose-escalation design; part two was a parallel group design using all three ATL-104 doses. Patients were followed up for 28 days post-treatment. Severity of signs and symptoms were assessed and used to calculate scores for standard toxicity rating scales (WHO, Western Consortium for Cancer Nursing Research (WCCNR)). Sixty-three patients were treated. Treatment with ATL-104 substantially reduced the median duration of severe oral mucositis (WHO grade 3 or 4) compared with placebo (median duration: ATL-104 groups 2 or 3 days, placebo 10.5 days). The effect of ATL-104 on the incidence of severe oral mucositis was inconclusive. Similar results were obtained using the WCCNR Scale. Adverse events (AEs) were predominantly mild or moderate in intensity. Gastrointestinal AE were most common.

We have demonstrated previously that cord blood CD133(+) cells isolated in the G(0) phase of the cell cycle are highly enriched for haematopoietic stem cell (HSC) activity, in contrast to CD133(+)G(1) cells. Here, we have analysed the phenotype and functional properties of this population in more detail. Our data demonstrate that a large proportion of the CD133(+)G(0) cells are CD38 negative (60.4%) and have high aldehyde dehydrogenase activity (75.1%) when compared with their CD133(+)G(1) counterparts (13.5 and 4.1%, respectively). This suggests that stem cell activity resides in the CD133(+)G(0) population. In long-term BM cultures, the CD133(+)G(0) cells generate significantly more progenitors than the CD34(+)G(0) population (P0.001) throughout the culture period. Furthermore, a comparison of CD133(+)G(0) versus CD133(+)G(1) cells revealed that multilineage reconstitution was obtained only in non-obese diabetic/SCID animals receiving G(0) cells. We conclude that CD133(+) cells in the quiescent phase of the cell cycle have a phenotype consistent with HSCs and are highly enriched for repopulating activity when compared with their G(1) counterparts. This cell population should prove useful for selection and manipulation in ex vivo expansion protocols.

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.

Recent evidence suggests a role for natural killer (NK) cells in the control of multiple myeloma. We show that expression of the NK cell receptor DNAM-1 (CD226) is reduced on CD56dim NK cells from myeloma patients with active disease compared with patients in remission and healthy controls. This suggested that this receptor might play a role in NK-myeloma interactions. The DNAM-1 ligands Nectin-2 (CD112) and the poliovirus receptor (PVR; CD155) were expressed by most patient myeloma samples analyzed. NK killing of patient-derived myelomas expressing PVR and/or Nectin-2 was DNAM-1 dependent, revealing a functional role for DNAM-1 in myeloma cell killing. In myeloma cell lines, cell surface expression of PVR was associated with low levels of NKG2D ligands, whereas cells expressing high levels of NKG2D ligands did not express PVR protein or mRNA. Furthermore, NK cell-mediated killing of myeloma cell lines was dependent on either DNAM-1 or NKG2D but not both molecules. In contrast, the natural cytotoxicity receptor NKp46 was required for the killing of all myeloma cell lines analyzed. Thus, DNAM-1 is important in the NK cell-mediated killing of myeloma cells expressing the cognate ligands. The importance of NKp46, NKG2D, and DNAM-1 in myeloma killing mirrors the differential expression of NK cell ligands by myeloma cells, reflecting immune selection during myeloma disease progression. [Cancer Res 2007;67(18):8444–9]

Increasing demand on the apheresis service makes efficient harvesting of peripheral blood stem cells (PBSCs) essential. A total of 168 adult patients with haematological malignancy were primed using low-moderate dose cyclophosphamide (1.5-3 g/m(2)) with G-CSF 5-10 microg/kg per day. Harvesting was booked and peripheral blood (PB) counts first checked between 6 and 10 days post-priming. One hundred and thirty (77%) patients harvested successfully (total harvest yieldor =2 x 10(6) CD34(+)/kg) and the median PBSC collection per procedure was 2.18 x 10(6)/kg (range 0.1-14.5). Only more lines of prior chemotherapy predicted failure to harvest in multivariate analysis (P=0.003). The PB CD34(+) cell count correlated significantly with harvest yield (r=0.8448, P0.0001). A PB CD34(+) countor =10/microl predicted a collection ofor =2 x 10(6)/kg (positive-predictive value of 61%, negative-predictive-value 100%). Patients first attending day 9 required significantly fewer visits to achieve a successful harvest than those first attending days 6-8 without increasing the risk of failure. No significant difference in failure rates, number of days attending and total harvest yield was found between days 9 and 10 attendees. Collection from day 9 may however enable higher target yields to be achieved. PB CD34(+) count monitoring should commence and harvesting booked from day 9 to optimize both the harvest and the efficiency of the PBSC harvesting service.

High-risk human papillomavirus (HPV) is a major causative agent of cervical cancer and the E6 and E7 genes encode the major HPV oncoproteins. The E7 protein from high-risk HPV types alters cell cycle progression and represses genes encoding components of the antigen-presentation pathway, suggesting a role for E7 in tumour immune evasion. We show that knockdown of E7 expression in HPV16- and HPV18-transformed cervical carcinoma cells by RNA interference increased expression of major histocompatibility complex (MHC) class I at the cell surface and reduced susceptibility of these cells to natural killer (NK) cells. Tetracycline-regulated induction of HPV16 E7 resulted in reduced expression of cell surface MHC class I molecules and increased NK cell killing. Our results suggest that, for HPV-associated malignancies, reduced MHC class I expression is the result of an active immune evasion strategy that has evolved to assist viral replication.

Natural-killer (NK) cells play an important role in recognizing and eliminating virally infected and transformed cells. To study this process, convenient assays for NK-cell function are required. Conventional NK-cell activity assays measure the release of 51Cr from prelabeled target cells following membrane disruption. This chapter describes nonradiometric assays for NK-cell killing of adenovirus-transformed human cells that can be applied to multiple cell samples using flow cytometry.

Expression of CUB domain containing protein 1 (CDCP1) is upregulated in carcinoma cells. We quantitated CDCP1 gene expression in matched normal colon and tumour tissue and compared the level of expression to other genes upregulated in colorectal tumourigenesis. Furthermore, we show that the CDCP1 gene generates two transcripts which are co-expressed in normal and matched tumour tissue as well as in the majority of cell lines analysed. However, intracellular localisation studies revealed that only one of these transcripts encodes a protein that is localised to the cell surface.

Activation of granzyme B, a key cytolytic effector molecule of natural killer (NK) cells, requires removal of an N-terminal pro-domain. In mice, cathepsin C is required for granzyme processing and normal NK cell cytolytic function, whereas in patients with Papillon-Lefèvre syndrome (PLS), loss-of-function mutations in cathepsin C do not affect lymphokine activated killer (LAK) cell function. Here we demonstrate that resting PLS NK cells do have a cytolytic defect and fail to induce the caspase cascade in target cells. NK cells from these patients contain inactive granzyme B, indicating that cathepsin C is required for granzyme B activation in unstimulated human NK cells. However, in vitro activation of PLS NK cells with interleukin-2 restores cytolytic function and granzyme B activity by a cathepsin C-independent mechanism. This is the first documented example of a human mutation affecting granzyme B activity and highlights the importance of cathepsin C in human NK cell function.

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.

Germ line mutations in the Adenomatous polyposis coli tumor suppressor gene cause a hereditary form of intestinal tumorigenesis in both mice and man. Here we show that in ApcMin/+ mice, which carry a heterozygous germ line mutation at codon 850 of Apc, there is progressive loss of immature and mature thymocytes from approximately 80 days of age with complete regression of the thymus by 120 days. In addition, ApcMin/+ mice show parallel depletion of splenic natural killer (NK) cells, immature B cells, and B progenitor cells in bone marrow due to complete loss of interleukin 7 (IL-7)-dependent B-cell progenitors. Using bone marrow transplantation experiments into wild-type recipients, we have shown that the capacity of transplanted ApcMin/+ bone marrow cells for T- and B-cell development appears normal. In contrast, although the ApcMin/+ bone marrow microenvironment supported short-term reconstitution with wild-type bone marrow, ApcMin/+ animals that received transplants subsequently underwent lymphodepletion. Fibroblast colony-forming unit (CFU-F) colony assays revealed a significant reduction in colony-forming mesenchymal progenitor cells in the bone marrow of ApcMin/+ mice compared with wild-type animals prior to the onset of lymphodepletion. This suggests that an altered bone marrow microenvironment may account for the selective lymphocyte depletion observed in this model of familial adenomatous polyposis. (Blood. 2004;103:1050-1058)

Unsuccessful stem cell remobilization for autologous transplantation is predicted by renal impairment and a stem cell yield 0.5 × 10 6 CD34 + cells/kg at first mobilization

The maps of the human immunoglobulin heavy-chain and kappa light-chain loci have recently been completed. We have now completed a map of the human lambda locus (IGL) located on chromosome 22q11.2. We mapped 52 V lambda genes from 10 V lambda families and 7 J lambda and C lambda genes on a 1140 kb contig constructed from eight YACs and 129 cosmid clones. The V lambda genes are arranged within 800 kb. Genes of the different V lambda families are organized in three clusters, V lambda II and III families (cluster A); V lambda I, V, VII and IX families (cluster B); V lambda IV, VI, VIII and X families (cluster C), in contrast to the dispersed organization of the different VH and V kappa families within the human VH and V kappa loci. We note that the most frequently used V lambda families (V lambda II and III) are proximal to the J lambda and C lambda genes. The VpreB gene, encoding part of the surrogate light chain, the GGT2 gene and the BCRL4 pseudogene were also mapped within the lambda locus.

We have previously described a strategy for integrating selectable marker genes into yeast artificial chromosomes (YACs) to facilitate their transfer into embryonic stem (ES) cells. Here we apply this technology to create mice carrying the core region of the human immunoglobulin (Ig) kappa light chain locus. A YAC was isolated which contains a 300 kb insert spanning three V kappa segments, the J kappa cluster, the C kappa region and extending downstream of the Kde element. After modification of this YAC to integrate the selectable neo marker gene, the YAC was introduced into ES cells by protoplast fusion. Several ES cell clones were obtained which appeared to harbor one complete copy of the YAC while retaining little or no other yeast DNA. The ES cells were injected into blastocysts and the chimaeric mice were shown to rearrange the introduced human light chain genes with the resultant production of antibodies containing human kappa light chains in the serum.