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A key unknown of the functional space in tumor immunity is whether CD4 T cells depend on intratumoral MHCII cancer antigen recognition. MHCII-expressing, antigen-presenting cancer-associated fibroblasts (apCAFs) have been found in breast and pancreatic tumors and are considered to be immunosuppressive. This analysis shows that antigen-presenting fibroblasts are frequent in human lung non-small cell carcinomas, where they seem to actively promote rather than suppress MHCII immunity. Lung apCAFs directly activated the TCRs of effector CD4 T cells and at the same time produced C1q, which acted on T cell C1qbp to rescue them from apoptosis. Fibroblast-specific MHCII or C1q deletion impaired CD4 T cell immunity and accelerated tumor growth, while inducing C1qbp in adoptively transferred CD4 T cells expanded their numbers and reduced tumors. Collectively, we have characterized in the lungs a subset of antigen-presenting fibroblasts with tumor-suppressive properties and propose that cancer immunotherapies might be strongly dependent on in situ MHCII antigen presentation.

In situ antigen presentation is required to sustain active proliferating CD4+ T cells in tumours and to help form memory CD8+ T cells, but the antigen presenting cells (APCs) and pathways involved remain elusive. Cancer associated fibroblasts (CAFs) are prominent stromal constituent of solid tumors. Current immunological dogma considers that CAFs facilitate tumour immune escape. A new subset of MHCII antigen presenting cancer-associated fibroblasts (apCAFs) has been described, but their function remains unknown. Here we report a previously unrecognized function of apCAFs in sustaining CD4+ T cells in primary human and murine lung tumours. In response to IFNγ and oxidative stress in tumours CAFs up-regulated MHCII. Fibroblast-specific targeted ablation of MHCII induced a hypometabolic hypoproliferative state in CD4+ T cells, which impacted MHCII and MHCI immunity, accelerating tumor growth. apCAFs directly presented MHCII-peptide (MCHIIp) complexes to activate the TCRs of CD4+ T cell. Highthrough-put profiling and blocking assays unveiled a novel CAF to T cell communication pathway via complement 1q binding on membrane C1qbp. Thus, apCAFs sustain anti-tumor CD4+ T cells via MHCIIp-TCR and C1q-C1qbp binding. Our studies pave the way to the design of novel immunotherapeutic strategies that will harness apCAFs to help sustain T cells inside solid tumours. Statement of significance This work challenges the immunological dogma that dominant physiological significance in tumour specific immunity comes only through professional APCs, leading to the design of novel MHCII fibroblast-directed strategies to sustain endogenous or adoptively transferred CD4+ T cells within solid tumors.

Summary Chimeric antigen receptor anti-CD19 (CAR19)-T cell immunotherapy-induced clinical remissions in CD19+ B cell lymphomas are often short lived. We tested whether CAR19-engineering of the CD1d-restricted invariant natural killer T (iNKT) cells would result in enhanced anti-lymphoma activity. CAR19-iNKT cells co-operatively activated by CD1d- and CAR19-CD19-dependent interactions are more effective than CAR19-T cells against CD1d-expressing lymphomas in vitro and in vivo. The swifter in vivo anti-lymphoma activity of CAR19-iNKT cells and their enhanced ability to eradicate brain lymphomas underpinned an improved tumor-free and overall survival. CD1D transcriptional de-repression by all-trans retinoic acid results in further enhanced cytotoxicity of CAR19-iNKT cells against CD19+ chronic lymphocytic leukemia cells. Thus, iNKT cells are a highly efficient platform for CAR-based immunotherapy of lymphomas and possibly other CD1d-expressing cancers., Graphical Abstract, Highlights • Bespoke protocol for CAR19-iNKT cell transduction and clinical scale expansion • Higher CAR19-iNKT than CAR19-T cell expandability and killing of CD19+CD1d+ targets • CAR19-iNKT cell reactivity potentiation by αGalCer and ATRA • Prolonged survival and brain lymphoma eradication of CAR19-iNKT cell-treated mice, Rotolo et al. show that anti-CD19 chimeric antigen receptor (CAR19)-engineered CD1d-restricted invariant NKT cells (iNKT) are more effective than CAR19-T cells against CD1d-expressing lymphomas, including those in the brain. De-repression of CD1d expression further enhances the anti-tumor efficacy of CAR19-iNKT.

We report that subunits of human nuclear proteasomes carry a previously unrecognised, constitutive posttranslational modification. Subunits with this modification are not visualised by SDS-PAGE, which is used in almost all denaturing protein gel electrophoresis. In contrast, CTAB-PAGE readily visualises such modified subunits. Thus, under most experimental conditions, with identical samples, SDS-PAGE yielded gel electrophoresis patterns for subunits of nuclear proteasomes which were misleading and strikingly different from those obtained with CTAB-PAGE. Initial analysis indicates a novel modification of a high negative charge with some similarity to polyADP-ribose, possibly explaining compatibility with (positively-charged) CTAB-PAGE but not (negatively-charged) SDS-PAGE and providing a mechanism for how nuclear proteasomes may interact with chromatin, DNA and other nuclear components.

Glycosphingolipids (GSLs) are essential constituents of cell membranes and lipid rafts and can modulate signal transduction events. The contribution of GSLs in osteoclast (OC) activation and osteolytic bone diseases in malignancies such as the plasma cell dyscrasia multiple myeloma (MM) is not known. Here, we tested the hypothesis that pathological activation of OCs in MM requires de novo GSL synthesis and is further enhanced by myeloma cell-derived GSLs. Glucosylceramide synthase (GCS) inhibitors, including the clinically approved agent N-butyl-deoxynojirimycin (NB-DNJ), prevented OC development and activation by disrupting RANKL-induced localization of TRAF6 and c-SRC into lipid rafts and preventing nuclear accumulation of transcriptional activator NFATc1. GM3 was the prevailing GSL produced by patient-derived myeloma cells and MM cell lines, and exogenous addition of GM3 synergistically enhanced the ability of the pro-osteoclastogenic factors RANKL and insulin-like growth factor 1 (IGF-1) to induce osteoclastogenesis in precursors. In WT mice, administration of GM3 increased OC numbers and activity, an effect that was reversed by treatment with NB-DNJ. In a murine MM model, treatment with NB-DNJ markedly improved osteolytic bone disease symptoms. Together, these data demonstrate that both tumor-derived and de novo synthesized GSLs influence osteoclastogenesis and suggest that NB-DNJ may reduce pathological OC activation and bone destruction associated with MM.

Multiple myeloma (MM) is an incurable tumor of the plasma cells, the terminally differentiated immunoglobulin secreting B lineage cells. The genetic make-up of MM has been extensively characterized but its impact on the biology of the disease is incomplete without more precise knowledge of the identity and functional role of cells with multiple myeloma propagating activity (MMPA). We review here recent data that link MMPA with myeloma clonotypic populations organized in a cellular hierarchy that mirrors normal B cell development and also with drug resistance and disease relapse. We further propose a conceptual framework which, with optimal use of recent technological advances in genomics and phenomics, could allow dissection of the cellular and molecular properties of cells with MMPA, drug resistance and in vivo relapse in an integrated and patient-specific manner. There is real hope that these approaches will significantly contribute to further improvements in disease control, overall survival, and possibly cure of patients with MM. Stem Cells 2015;33:3205–3211

Swap70 functions as a guanine nucleotide exchange factor for Rac and RhoA regulating F-actin cytoskeletal rearrangements and playing a crucial role in mammalian cell activation, migration, adhesion and invasion. Here we show that the zebrafish orthologue, Swap70b, is required for convergent and extension cell movement during gastrulation. Swap70b morphants exhibited broader and shorter body axis but cell fate specification appeared normal. While ectopic Swap70b expression robustly rescued Wnt11 morphants, RhoA overexpression was sufficient to rescue Swap70b morphants, establishing Swap70b as a novel member of the non-canonical Wnt/PCP pathway downstream of Wnt11 and upstream of RhoA. This is distinct from the related Def6a protein that acts downstream of Wnt5b. Def6a/Swap70b morphants resemble Ppt/Slb double mutant embryos suggesting that Swap70b and Def6a delineate Wnt11 and Wnt5b signalling pathways.

Introduction: Development of mature B cells depends upon the expression of a functional B cell receptor (BCR) and of its immunoglobulin (Ig) heavy (H) and light (L) chains. The molecular hallmark of B cell development, somatic recombination of the genes that encode the IgH (V, D and J) and IgL (V and J) chains, takes place in B cells in primary B lymphopoiesis sites throughout development (i.e. fetal liver (FL), fetal bone marrow (FBM) and adult BM). However, the spatiotemporal relationship between the IgH repertoire in FL with that in FBM, and the impact of the fetal Ig repertoire on the long-term repertoire present in post-natal life, as well as the link between this and the development of disease, are largely unknown. Aim: To gain insights into the ontogeny of the human innate B cell repertoire by performing high resolution analysis of the IgH-Cmu repertoire of human FL, FBM and post-natal B cells. Methods: CD34negCD19pos B cells from human 2nd trimester FL and FBM, child peripheral blood (cPB) and adult peripheral blood (aPB) were isolated by flow sorting. For initial assessment of the IgM repertoire ontogeny in FL and FBM B cells, we performed CDR3 fragment length analysis of VH1-VH6 families in the B cells. To gain further insights into the ontogeny of IgH diversification, we employed 454 next generation sequencing (NGS)-based analysis of the IgVH-Cmu mRNA repertoire in FL, FBM and postnatal B cells. In total, 20 libraries generated from 17 individual, flow-sorted CD34negCD19pos B cell samples were sequenced: 5 FL (4 performed in independent duplicate libraries; GA 14-18 weeks), 3 FBM (GA 13-17 weeks), 3 cPB (age 4mo-4yrs) and 5 aPB (age 29-53 yrs) samples. Bioinformatic analysis was performed with the ARResT/Interrogate immunoprofiler, IMGT tools, and appropriate statistics. Results: Spectratyping and NGS data showed that the molecular mechanisms responsible for VDJ recombination-dependent repertoire diversification, such as convergent recombination, are active in early B cell development and as efficient as postnatal B cells in both FL and FBM. Comparably diversified B cell lymphopoiesis exists contemporaneously in FL and FBM. However, analysis of the top 100 most abundant clonotypes in each developmental stage showed that their mean abundance in FBM B cells was 10 times lower (0.12%) than in FL B cells (1.2%; p Conclusions: The lack of mature, antigen-responsive B cells in FBM and the pronounced clonotypic expansions in FL suggest that FL is the main source of IgM natural immunity during the 2nd trimester. Our results are consistent with persisting fetal IgM+ B cells being an important source of the natural IgM repertoire also in adult life. Further, the origins of specific stereotypic IgM+ BCR associated with CLL, can be traced back to fetal B cell lymphopoiesis, suggesting that persisting fetal B cells can be subject to malignant transformation late in life. Overall, these novel data provide unique insights into the ontogeny of physiological and malignant B lymphopoiesis that spans the human lifetime. Disclosures No relevant conflicts of interest to declare.

The 40-fold increase in childhood megakaryocyte-erythroid and B-cell leukemia in Down syndrome implicates trisomy 21 (T21) in perturbing fetal hematopoiesis. Here, we show that compared with primary disomic controls, primary T21 fetal liver (FL) hematopoietic stem cells (HSC) and megakaryocyte-erythroid progenitors are markedly increased, whereas granulocyte-macrophage progenitors are reduced. Commensurately, HSC and megakaryocyte-erythroid progenitors show higher clonogenicity, with increased megakaryocyte, megakaryocyte-erythroid, and replatable blast colonies. Biased megakaryocyte-erythroid–primed gene expression was detected as early as the HSC compartment. In lymphopoiesis, T21 FL lymphoid-primed multipotential progenitors and early lymphoid progenitor numbers are maintained, but there was a 10-fold reduction in committed PreproB-lymphoid progenitors and the functional B-cell potential of HSC and early lymphoid progenitor is severely impaired, in tandem with reduced early lymphoid gene expression. The same pattern was seen in all T21 FL samples and no samples had GATA1 mutations. Therefore, T21 itself causes multiple distinct defects in FL myelo- and lymphopoiesis.

In Down syndrome (DS), trisomy 21 (T21) causes perturbation of fetal liver (FL) hematopoiesis leading to expansion of megakaryocyte-erythroid (MK-E) progenitors. On this background, FL hematopoietic stem/progenitor cells (HSPC) may acquire GATA1 gene mutations which usually present as a neonatal preleukemic condition, Transient Myeloproliferative Disorder (TMD). TMD develops only in fetal life and most cases spontaneously regress after birth, suggesting unique features of the T21 fetal microenvironment may be important in driving both abnormal DS FL hematopoiesis and mutant GATA1 clones in TMD. Previous work showed activation of insulin-like growth factor (IGF) signalling in TMD and DS-AMKL and fetal-specific IGF dependence of murine MK development implicating T21 in altered fetal IGF signalling. To investigate the role of IGF signalling in fetal MK-E development in DS FL without GATA1 mutations, we performed gene expression profiling (GEP) of DS FL (n=5) and normal FL CD34+ cells (n=3). GSEA showed significant enrichment of IGF targets. We therefore measured expression of the principal IGF receptor, IGF1R, on DS FL HSPC (n=6). Surface IGF1R was uniformly expressed on all DS FL HSPC subpopulations. However, there was no difference in IGF1R surface or gene expression between DS and normal FL HSPC (n=8) suggesting that increased IGF1R expression on TMD and DS-AMKL cells is an early event and may reflect their fetal rather than T21 origin. To determine if the enrichment of IGF targets in DS FL HSPC might reflect increased IGF production in the FL microenvironment, we compared IGF expression in DS FL mesenchymal stromal cells (MSC)(n=4) and hepatocytes with normal FL MSC (n=4). Both DS and normal FL MSC expressed large amounts of IGF2, but little or no IGF1 with no difference between DS and normal MSC. Similarly, IGF2 was expressed by IHC at similar levels in both DS and normal fetal hepatocytes and MSC whereas IGF1 was barely detectable. Normal and DS fetal bone marrow (BM) MSC also expressed high levels of IGF2, but not IGF1 in contrast to adult BM MSC. Thus, IGF2 is the main IGF produced in the human fetal hematopoietic environment. Consistent with a specific role in DS FL hematopoiesis, IGF2 caused marked proliferation of DS FL clonogenic MK and MK-E cells (n=3) and stimulated MK proliferation in liquid culture while in normal FL, IGF2 stimulated EPO-independent BFU-E and Pre-BFU-E but had no effect on MK cells. Since differential effects of IGF2 on DS/normal FL HSPC might be due to altered expression of the Insulin Receptor (InsR), which binds IGF2 with low affinity, we compared InsR surface and gene expression in DS and normal FL HSPC but found no significant difference. IGF2 also binds to IGF2R, a negative regulator of IGF2, however there was no difference in IGF2R expression between DS and normal FL HSPC. Co-culture of normal FL CD34+ cells with DS or normal FL MSC (n=3) promoted MK and erythroid cell proliferation and differentiation. Since the effect of DS FL MSC on normal FL CD34+ cells was significantly greater (~4-fold) than that of normal FL MSC, the data above suggest this is unlikely to be due to IGF2 alone. To investigate whether T21 alters the MSC secretome, we performed GEP of DS FL MSC (n=3) and compared this with normal FL MSC (n=5). Both normal FL and DS FL expressed known hematopoietic cytokine genes, including SCF, CXCL12, G-CSF, M-CSF, Flt3 and IL-6, as well as IGF2 but not IGF1, EPO or TPO. Of the top 80 differentially expressed genes (Fold Change>2), ~40% encode secreted proteins, including several which affect IGF availability. Conclusion: T21 perturbs FL hematopoiesis through both HSPC-intrinsic mechanisms, including changes in IGF responsiveness, and through alterations to the FL microenvironment. Disclosures No relevant conflicts of interest to declare.

Despite the therapeutic advances that followed the introduction of new immunomodulatory drugs and proteasome inhibitors, multiple myeloma (MM) remains an incurable malignancy and eventually all patients develop and succumb to chemo-refractory disease. The recently developed bromodomain and extra terminal (BET) protein inhibitors are novel agents targeting the acetyl-binding pockets of the BET family proteins BRD2-4 and BRDT. BET proteins activate transcription through their ability to bind to acetyl-modified lysine residues of histone tails, thereby serving as chromatin scaffolds that recruit the P-TEFb and PAFc1 complexes to Polymerase II (RNA Pol II), ensuring transcriptional initiation and elongation. In preclinical models, two classes of BET inhibitors, benzodiazepines (e.g JQ1) and quinolones (e.g. I-BET151), have been shown to have significant anti-proliferative activity against a variety of hematologic tumours. However translation of these data to molecules suitable for clinical development has yet to be disclosed. Herein, we tested the anti-myeloma activity and extended the mechanistic insights on two BET inhibitors: the chemical probe molecule I-BET151 and I-BET762, an orally active benzodiazepine suitable for clinical development. I-BET151 was tested in vitro in 6 myeloma cell lines (MMCL) with cytogenetic profiles representative of the most common translocations found in MM. I-BET151 induces apoptosis and cell cycle arrest in all MMCL in a time-dependent manner, with IC50 ranging from 133nM to 411nM at 72hrs. With the exception of KMS11 cells, IC50 was similar in stroma-free conditions and in co-culture with MS5 stromal cells. Similarly, I-BET151 induces apoptosis and cell cycle arrest in primary MM cells (n=4) cultured in the presence of IL-6 and stroma. In a subcutaneous MM mouse model, as compared to placebo, treatment with I-BET151 30mg/Kg/day i.p for 21 days resulted in 4-5 fold reduction in tumour size (p In conclusion, I-BET151 and I-BET762 show potent anti-myeloma activity in vitro and in vivo. I-BET151 inhibition of BRD2-4 binding mediates MYC transcriptional silencing and cell cycle arrest, but MYC-independent mechanism are also likely to mediate the I-BET biological effects in MM. Our data is the first example of an orally active BET inhibitor significantly delaying MM progression in vivo and provides strong rationale for clinical testing in phase I/II trials. Disclosures: Tough: GlaxoSmithKline: Employment. Smithers:GlaxoSmithKline: Employment. Bassil:GlaxoSmithKline: Employment. Chapman:GlaxoSmithKline: Employment. Harker:GlaxoSmithKline: Employment. Barbash:GlaxoSmithKline: Employment. Tummino:GlaxoSmithKline: Employment. Al-Mahdi:GlaxoSmithKline: Employment. Haynes:GlaxoSmithKline: Employment. Cutler:GlaxoSmithKline: Employment. Le:GlaxoSmithKline: Employment. Witherington:GlaxoSmithKline: Employment. Parr:GlaxoSmithKline: Employment. Prinjha:GlaxoSmithKline: Employment.