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Intratumoral regulatory T (Treg) cell abundance associates with diminished antitumor immunity and poor prognosis in human cancers. Recent work demonstrates that CD25, the high-affinity receptor subunit for interleukin (IL)-2, is a selective target for Treg depletion in mouse and human malignancies; however, anti-human CD25 antibodies have failed to deliver clinical responses against solid tumors due to bystander IL-2 receptor signaling blockade on effector T cells, which limits their antitumor activity. Here we demonstrate potent single-agent activity of anti-CD25 antibodies optimized to deplete Treg cells, while preserving IL-2-STAT5 signaling on effector T cells and show synergy with immune checkpoint blockade in vivo. Pre-clinical evaluation of an anti-human CD25 (RG6292) antibody with equivalent features demonstrates, in both nonhuman primates and humanized mouse models, efficient Treg cell depletion with no overt immune-related toxicities. Our data support the clinical development of RG6292 and evaluation of new combination therapies incorporating non-IL-2-blocking anti-CD25 antibodies in clinical studies. Quezada and colleagues develop improved anti-CD25 antibodies that preserve IL-2 signaling and enhance single-agent antitumor immunity and immunotherapy through specific and efficient Treg cell depletion.

Purpose: Immune dysregulation is described in multiple myeloma. While preclinical models suggest a role for altered T-cell immunity in disease progression, the contribution of immune dysfunction to clinical outcomes remains unclear. We aimed to characterize marrow-infiltrating T cells in newly diagnosed patients and explore associations with outcomes of first-line therapy. Experimental Design: We undertook detailed characterization of T cells from bone marrow (BM) samples, focusing on immune checkpoints and features of immune dysfunction, correlating with clinical features and progression-free survival. Results: We found that patients with multiple myeloma had greater abundance of BM regulatory T cells (Tregs) which, in turn, expressed higher levels of the activation marker CD25 compared with healthy donors. Patients with higher frequencies of Tregs had shorter PFS and a distinct Treg immune checkpoint profile (increased PD-1, LAG-3) compared with patients with lower frequencies of Tregs. Analysis of CD4 and CD8 effectors revealed that low CD4effector (CD4eff):Treg ratio and increased frequency of PD-1–expressing CD4eff cells were independent predictors of early relapse over and above conventional risk factors, such as genetic risk and depth of response. Ex vivo functional analysis and RNA sequencing revealed that CD4 and CD8 cells from patients with greater abundance of CD4effPD-1+ cells displayed transcriptional and secretory features of dysfunction. Conclusions: BM-infiltrating T-cell subsets, specifically Tregs and PD-1–expressing CD4 effectors, negatively influence clinical outcomes in newly diagnosed patients. Pending confirmation in larger cohorts and further mechanistic work, these immune parameters may inform new risk models, and present potential targets for immunotherapeutic strategies.

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies., Graphical Abstract, Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Burnett et al. immunized humanized mice with different diverse sarbecovirus RBDs to elicit antibodies targeting conserved sites. Non-neutralizing cross-reactive antibodies targeting the conserved class 4 epitope were readily elicited. Neutralizing ability was reserved only for antibodies binding this conserved supersite through an elongated CDRH3 that obstructed ACE2-RBD interactions.

Antibodies against coronavirus spike protein potently protect against infection and disease, but whether such protection can be extended to variant coronaviruses is unclear. This is exemplified by a set of iconic and well-characterized monoclonal antibodies developed after the 2003 SARS outbreak, including mAbs m396, CR3022, CR3014 and 80R, which potently neutralize SARS-CoV-1, but not SARS-CoV-2. Here, we explore antibody engineering strategies to change and broaden their specificity, enabling nanomolar binding and potent neutralization of SARS-CoV-2. Intriguingly, while many of the matured clones maintained specificity of the parental antibody, new specificities were also observed, which was further confirmed by X-ray crystallography and cryo-electron microscopy, indicating that a limited set of VH antibody domains can give rise to variants targeting diverse epitopes, when paired with a diverse VL repertoire. Our findings open up over 15 years of antibody development efforts against SARS-CoV-1 to the SARS-CoV-2 field and outline general principles for the maturation of antibody specificity against emerging viruses.

Antibodies against coronavirus spike protein potently protect against infection and disease, however it remains unclear if such protection can be extended to variant coronaviruses. This is exemplified by a set of iconic and well-characterized monoclonal antibodies developed after the 2003 SARS outbreak including mAbs m396, CR3022, CR3014 and 80R, which potently neutralize SARS-CoV-1, but not SARS-CoV-2. Here we explore antibody maturation strategies to change and broaden their specificity, enabling potent binding and neutralization of SARS-CoV-2. Using targeted mutagenesis as well as light chain shuffling on phage, we identified variants with considerably increased affinity and neutralization potential. The most potent antibody, derived from the NIH-developed mAb m396, neutralized live SARS-CoV-2 virus with a half-maximal inhibitory concentration (IC50) of 160 ng/ml. Intriguingly, while many of the matured clones maintained specificity of the parental antibody, new specificities were also observed, which was further confirmed by X-ray crystallography and cryo-electron microscopy, indicating that a limited set of antibodies can give rise to variants targeting diverse epitopes. Our findings open up over 15 years of antibody development efforts against SARS-CoV-1 to the SARS-CoV-2 field and outline general principles for the maturation of antibody specificity against emerging viruses.

The retinal basal glia (RBG) is a group of glia that migrates from the optic stalk into the third instar larval eye disc while the photoreceptor cells (PR) are differentiating. The RBGs are grouped into three major classes based on molecular and morphological characteristics: surface glia (SG), wrapping glia (WG) and carpet glia (CG). The SGs migrate and divide. The WGs are postmitotic and wraps PR axons. The CGs have giant nucleus and extensive membrane extension that each covers half of the eye disc. In this study, we used lineage tracing methods to determine the lineage relationships among these glia subtypes and the temporal profile of the lineage decisions for RBG development. We found that the CG lineage segregated from the other RBG very early in the embryonic stage. It has been proposed that the SGs migrate under the CG membrane, which prevented SGs from contacting with the PR axons lying above the CG membrane. Upon passing the front of the CG membrane, which is slightly behind the morphogenetic furrow that marks the front of PR differentiation, the migrating SG contact the nascent PR axon, which in turn release FGF to induce SGs’ differentiation into WG. Interestingly, we found that SGs are equally distributed apical and basal to the CG membrane, so that the apical SGs are not prevented from contacting PR axons by CG membrane. Clonal analysis reveals that the apical and basal RBG are derived from distinct lineages determined before they enter the eye disc. Moreover, the basal SG lack the competence to respond to FGFR signaling, preventing its differentiation into WG. Our findings suggest that this novel glia-to-glia differentiation is both dependent on early lineage decision and on a yet unidentified regulatory mechanism, which can provide spatiotemporal coordination of WG differentiation with the progressive differentiation of photoreceptor neurons.

Background Effective anti-tumour immunity requires cancer antigen expression, but persistent antigen exposure in chronic viral infections and autoimmunity has a detrimental effect on immune function. This is associated with a decline of early differentiated T cell populations in favour of later differentiated, dysfunctional subsets, resulting in an unfavourable skewing of the immune landscape. It is unknown whether this occurs locally within the antigen rich tumour microenvironment, driving immune failure. Materials and Methods We combined tumour infiltrating lymphocyte (TIL) high dimensional flow cytometry, bulk exome and RNA sequencing data from multiregional samples obtained from surgically resected tumours of treatment naive patients with non-small cell lung cancer (NSCLC) amongst the first 100 recruited to the prospective, UK-wide lung TRACERx study. Clonal relationship between T cell populations was determined by T cell receptor (TCR) sequencing. We additionally analysed publically available single T cell RNA sequencing data and bulk RNA sequencing data within TCGA. Results T cell differentiation skewing (TDS) occurred amongst TILs in association with tumour mutational burden (TMB). Surprisingly, this was most evident within the CD4 compartment that had a greater abundance of central memory cells expressing the key transcription factor TCF7. Amongst CD4 cells, loss of a PD1-CCR7+ T central memory population was accompanied by gain in abundance of PD1+ populations with exhausted (CD57-ICOShiCTLA4hi) and terminally differentiated effector (CD57+Eomes+) features. TCR sequencing revealed early and dysfunctional differentiated populations to be clonally related and CDR3 clustering analysis showed greater similarity of sequences shared vs. non-shared between subsets, consistent with an antigen driven differentiation process. Similar patterns were observed within the CD8 compartment. Identification of these subsets within single T cell RNA sequencing data revealed shared and distinct functional regulators, suggesting the enhanced effector capability of early compared to dysfunctionally differentiated populations. A validated transcriptional signature of TDS generated using TRACERx samples with paired flow cytometry and RNA sequencing data reflected loss of gene expression downstream of TCF7, and predicted worse survival within TRACERx and multiple TCGA cohorts including lung adenocarcinoma (LUAD). Conclusions Our finding support a model of neoantigen driven T cell differentiation within the tumour microenvironment that drives the depletion of progenitor-like cells and gain in abundance of dysfunctional subsets, resulting in a loss of immune fitness. Our analysis of transcriptomic data elucidates potential regulatory mechanisms and therapeutic targets within the subsets identified. Disclosure Information E. Ghorani: None. J. Reading: None. J. Henry: None. M. Robert de Massy: None. R. Rosenthal: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Achilles Therapeutics. F. Consultant/Advisory Board; Modest; Achilles Therapeutics. V. Turati: None. A. Furness: None. A. Ben Aissa: None. S. Kumar Saini: None. S. Ramskov: None. A. Georgiou: None. M. Vila De Mucha: None. I. Uddin: None. T. Ronel: None. R. Salgado: None. T. Lund: None. J. Herrero: None. T. Enver: None. S. Hadrup: None. A. Hackshaw: None. K. Peggs: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Achilles Therapeutics. N. McGranahan,: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Achilles Therapeutics. F. Consultant/Advisory Board; Modest; Achilles Therapeutics. B. Chain: None. C. Swanton: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Pfizer, AstraZeneca, BMS, Roche–Ventana and Boehringer Ingelheim. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; ApoGen Biotechnologies, Epic Bioscience and GRAIL, and has stock options in and is co-founder of Achilles Therapeutics. F. Consultant/Advisory Board; Modest; Pfizer, Novartis, GlaxoSmithKline, MSD, BMS, Celgene, AstraZeneca, Illumina, Genentech, Roche–Ventana, GRAIL, Medicxi and the Sarah Cannon Research Institute. S. Quezada: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Achilles Therapeutics.

Before squamous cell lung cancer develops, precancerous lesions can be found in the airways. From longitudinal monitoring, we know that only half of such lesions become cancer, whereas a third spontaneously regress. Although recent studies have described the presence of an active immune response in high-grade lesions, the mechanisms underpinning clinical regression of precancerous lesions remain unknown. Here, we show that host immune surveillance is strongly implicated in lesion regression. Using bronchoscopic biopsies from human subjects, we find that regressive carcinoma in situ lesions harbor more infiltrating immune cells than those that progress to cancer. Moreover, molecular profiling of these lesions identifies potential immune escape mechanisms specifically in those that progress to cancer: antigen presentation is impaired by genomic and epigenetic changes, CCL27–CCR10 signaling is upregulated, and the immunomodulator TNFSF9 is downregulated. Changes appear intrinsic to the carcinoma in situ lesions, as the adjacent stroma of progressive and regressive lesions are transcriptomically similar. Significance: Immune evasion is a hallmark of cancer. For the first time, this study identifies mechanisms by which precancerous lesions evade immune detection during the earliest stages of carcinogenesis and forms a basis for new therapeutic strategies that treat or prevent early-stage lung cancer. See related commentary by Krysan et al., p. 1442. This article is highlighted in the In This Issue feature, p. 1426

Before squamous cell lung cancer develops, pre-cancerous lesions can be found in the airways. From longitudinal monitoring, we know that only half of such lesions become cancer, whereas a third spontaneously regress. While recent studies have described the presence of an active immune response in high-grade lesions, the mechanisms underpinning clinical regression of pre-cancerous lesions remain unknown. Here, we show that host immune surveillance is strongly implicated in lesion regression. Using bronchoscopic biopsies from human subjects, we find that regressive carcinoma in-situ lesions harbour more infiltrating immune cells than those that progress to cancer. Moreover, molecular profiling of these lesions identifies potential immune escape mechanisms specifically in those that progress to cancer: antigen presentation is impaired by genomic and epigenetic changes, TGF-beta signalling is overactive, and the immunomodulator TNFSF9 is downregulated. Changes appear intrinsic to the CIS lesions as the adjacent stroma of progressive and regressive lesions are transcriptomically similar. This study identifies mechanisms by which pre-cancerous lesions evade immune detection during the earliest stages of carcinogenesis and forms a basis for new therapeutic strategies that treat or prevent early stage lung cancer.

Summary Inflammation triggers the differentiation of Ly6Chi monocytes into microbicidal macrophages or monocyte-derived dendritic cells (moDCs). Yet, it is unclear whether environmental inflammatory cues control the polarization of monocytes toward each of these fates or whether specialized monocyte progenitor subsets exist before inflammation. Here, we have shown that naive monocytes are phenotypically heterogeneous and contain an NR4A1- and Flt3L-independent, CCR2-dependent, Flt3+CD11c−MHCII+PU.1hi subset. This subset acted as a precursor for FcγRIII+PD-L2+CD209a+, GM-CSF-dependent moDCs but was distal from the DC lineage, as shown by fate-mapping experiments using Zbtb46. By contrast, Flt3−CD11c−MHCII−PU.1lo monocytes differentiated into FcγRIII+PD-L2−CD209a−iNOS+ macrophages upon microbial stimulation. Importantly, Sfpi1 haploinsufficiency genetically distinguished the precursor activities of monocytes toward moDCs or microbicidal macrophages. Indeed, Sfpi1+/− mice had reduced Flt3+CD11c−MHCII+ monocytes and GM-CSF-dependent FcγRIII+PD-L2+CD209a+ moDCs but generated iNOS+ macrophages more efficiently. Therefore, intercellular disparities of PU.1 expression within naive monocytes segregate progenitor activity for inflammatory iNOS+ macrophages or moDCs., Graphical Abstract, Highlights • Murine Ly6ChiCD115+ monocytes are heterogeneous • DC-related genes (Cd209a and MHCII) are expressed in a subset of FcγRIII+ monocytes • GM-CSF-dependent CD209a+ moDCs are generated by FcγRIII+CD209a+MHCII+ monocytes • iNOS+ macrophages are generated by FcγRIII+CD209a−MHCII− monocytes, Monocytes can differentiate into multiple progenies during inflammation. Here, Menezes et al. show that monocytes from naive mice are heterogeneous and contain distinct precursor subsets giving rise to iNOS+ inflammatory macrophages or GM-CSF-induced CD209a+ monocyte-derived dendritic cells.

Photoreceptor (PR) axons project from the retina to the optic lobe in brain and form a precise retinotopic map in the Drosophila visual system. Yet the role of retinal basal glia in the retinotopic map formation is not previously known. We examined the formation of the retinotopic map by marking single PR pairs and following their axonal projections. In addition to confirming previous studies that the spatial information is preserved from the retina to the optic stalk and then to the optic lamina, we found that the young PR R3/4 axons transiently overshoot and then retract to their final destination, the lamina plexus. We then examined the process of wrapping glia (WG) membrane extension in the eye disc and showed that the WG membrane extensions also follow the retinotopic map. We show that the WG is important for the proper spatial distribution of PR axons in the optic stalk and lamina, suggesting an active role of wrapping glia in the retinotopic map formation.

Neurons and glia interact reciprocally. Glia perform many important functions in the development and proper functioning of the nervous system throughout different stages of life. Neurons also affect the development and function of glia. Neurodegenerative diseases are usually late-onset, progressive, and affect specific parts of the nervous system. Many neurodegenerative disorders have been extensively studied. However, the majority of the studies have focused on the events that occur in neurons. The events that occur in glia, and whether and how glia participate in the pathogenesis of these diseases, have not been as well studied. In this review, we will focus on how the fruit fly Drosophila melanogaster has been used as a model to study neuron-glia interactions in neurodegenerative disorders. We discuss how glia are affected in these models of human neurodegenerative disorders and how glia contribute to their pathogenesis. These studies have provided important insight into the mechanisms of diverse neurodegenerative disorders, and have suggested possibilities for early diagnosis.

Expansion of poly-glutamine (polyQ) stretches in several proteins has been linked to neurodegenerative diseases. The effects of polyQ-expanded proteins on neurons have been extensively studied, but their effects on glia remain unclear. We found that expression of distinct polyQ proteins exclusively in all glia or specifically in the blood-brain barrier (BBB) and blood-retina barrier (BRB) glia caused cell-autonomous impairment of BBB/BRB integrity, suggesting that BBB/BRB glia are most vulnerable to polyQ-expanded proteins. Furthermore, we also found that BBB/BRB leakage in Drosophila is reflected in reversed waveform polarity on the basis of electroretinography (ERG), making ERG a sensitive method to detect BBB/BRB leakage. The polyQ-expanded protein Atxn3-84Q forms aggregates, induces BBB/BRB leakage, restricts Drosophila lifespan and reduces the level of Repo (a pan-glial transcriptional factor required for glial differentiation). Expression of Repo in BBB/BRB glia can rescue BBB/BRB leakage, suggesting that the reduced expression of Repo is important for the effect of polyQ on BBB/BRB impairment. Coexpression of the chaperon HSP40 and HSP70 effectively rescues the effects of Atxn3-84Q, indicating that polyQ protein aggregation in glia is deleterious. Intriguingly, coexpression of wild-type Atxn3-27Q can also rescue BBB/BRB impairment, suggesting that normal polyQ protein may have a protective function.

5D images of engrailed (en) and eye gone (eyg) gene expressions during the course of the eye-antenna disc primordium (EADP) formation of Drosophila embryos from embryonic stages 13 through 16 were recorded via light sheet microscopy and analyzed to reveal the cell dynamics involved in the development of the EADP. Detailed analysis of the time-lapsed images revealed the process of EADP formation and its invagination trajectory, which involved an inversion of the EADP anterior-posterior axis relative to the body. Furthermore, analysis of the en-expression pattern in the EADP provided strong evidence that the EADP is derived from one of the en-expressing head segments.

Introduction: High dose therapy and autologous stem cell transplant (ASCT) remain standard first line therapy for young fit patients with multiple myeloma (MM). Beyond cytoreduction, ASCT may also alter the bone marrow (BM) immune environment to augment anti-tumour immune responses. A more precise understanding of the immune microenvironment post ASCT and its relationship with clinical outcomes may identify biomarkers to refine prognostication and provide opportunity for therapeutic intervention. Aim: We aimed to define T cell subsets and expression of co-activating and co-inhibitory proteins in the BM early post ASCT, and explore associations with clinical outcomes. Methods: BM samples were obtained at 100 days (D100) post ASCT from 61 MM patients, and 15 healthy donors (HD). BM mononuclear cells (MNCs) were stained for surface (CD3, CD4, CD8, PD-1, LAG-3, and ICOS) and intracellular markers (GzmB, Ki-67, CTLA-4, and FoxP3). Data were acquired on a BD LSR Fortessa and analysed using FlowJo. Progression free survival (PFS) was defined from ASCT as per IMWG criteria, and patient groups were compared using Mann-Whitney U test. Adverse risk genetics was defined as t(4;14), t(14;16) or del(17p). Results: Median age of patients at ASCT was 58yrs (36-71), 70.5% were male. Median follow up was 17 months (3-54). At diagnosis, 36.1% were ISS stage I, 8.2% had extramedullary disease, and 14.7% had adverse risk genetics. As induction therapy, 96.7% received a proteasome inhibitor, 45.9% an immunomodulatory drug, 54.1% both, and 21.3% received post ASCT maintenance/consolidation. Median PFS was 24 months. At D100, 24.6% had achieved CR, 47.5% VGPR, 21.3% PR, 4.9% SD and none had PD. Improved PFS was found in patients with deeper response (CR/VGPR vs. rest, p=0.003), and with ISS stage I vs II/III (p=0.013). There was a trend for improved PFS with standard risk genetics (p=0.088 cf high risk). MM BM contained lower frequencies of CD3 and CD4 T cells compared to HD (CD3, 28% of live MNCs, vs 41.2%, p=0.012; CD4, 4.1% vs 14.0%, p Immune checkpoint proteins were highly expressed on BM T cell subsets in post ASCT compared to HD. CD4eff in MM patients expressed higher frequency of PD-1, LAG-3, ICOS, CTLA-4, GzmB, and Ki-67 (p Clinical outcomes were not influenced by T cell subsets (CD3, CD4eff, CD8, and Treg) but there was an inverse association with co-inhibitory receptor expression. High frequency (>median) of LAG-3 on CD4eff and CD8, and Tregs was associated with shorter PFS (median 18mo vs 53, p=0.0028; 20mo vs 53, p=0.0057; 20mo vs 53, p=0.046 respectively). Further, patients with high frequency of PD-1 on CD4eff and Ki-67 on CD8 T cells had shorter PFS (median 18mo vs 36, p=0.042 and 18mo vs unreached, p=0.019 respectively). A multivariate Cox regression model was built to include ISS stage, depth of response at D100, and the immune phenotypes identified to correlate with PFS. In this model, high frequency of PD-1 on CD4eff retained independent prognostic value, along with ISS stage and CD8 Ki67+ T cells. Conclusion: We present data indicating that high frequency of PD-1+ CD4eff and Ki67+ CD8 T cells post ASCT associate with shorter PFS in MM patients. Pending confirmation in larger cohorts, these data reveal potential immune biomarkers for poor outcomes, and prompt deeper phenotypic analysis and mechanistic studies to uncover the immune drivers of sustained remissions post ASCT. Disclosures Lee: Autolus Therapeutics: Equity Ownership, Research Funding. Yong:Takeda: Research Funding, Speakers Bureau; Autolus: Consultancy; Amgen: Research Funding, Speakers Bureau; Janssen: Speakers Bureau; Sanofi: Speakers Bureau.

2590 Background: CD4 T helper cells are key orchestrators of immunity in states of persistent antigen exposure. In chronic viral infection, loss of immune control is associated with CD4 differentiation skewing (CD4ds) resulting from decline of early progenitors and gain in abundance of exhausted and terminally differentiated subsets. Here, we set out to identify whether a similar process occurs within the tumour microenvironment, contributing to immune dysfunction. Methods: Multiregional samples of tumour and non-tumour lung tissue from patients with untreated, surgically resected non-small cell lung cancer (NSCLC) within the first 100 recruited to the prospective lung TRACERx study were analysed by high dimensional flow cytometry of tumour infiltrating lymphocytes (TILs) and paired bulk tumour exome and RNA sequencing. We additionally reanalysed publically available single T cell and bulk tumour RNA sequencing from patients with NSCLC. Results: Unsupervised clustering and dimension reduction revealed a heterogenous landscape of CD4 TILs, with evidence of CD4ds in association with tumour mutational burden. Loss of PD1-CCR7+ T central memory enriched early differentiated cells was accompanied by gain in abundance of PD1+ populations with exhausted (CD57-ICOShiCTLA4hi) and terminally differentiated effector (CD57+Eomes+) phenotypes. Further characterisation of these populations by single cell RNA sequencing revealed differential expression of key genes involved in transcriptional regulation, co-inhibition and co-stimulatory pathways. A validated gene signature of CD4ds was associated with worse outcomes in TRACERx and TCGA cohorts. Conclusions: Our findings reveal remodelling of the CD4 differentiation landscape in association with tumour genomic characteristics, underscoring how mutational burden in the context of chronic stimulation may lead to loss of immune fitness and elucidating key regulatory pathways in potentially clinically relevant CD4 subsets.

Anti–CTLA-4 antibody induces selective depletion of T reg cells within tumor lesions in a manner that is dependent on the presence of Fc gamma receptor-expressing macrophages within the tumor microenvironment., Treatment with monoclonal antibody specific for cytotoxic T lymphocyte–associated antigen 4 (CTLA-4), an inhibitory receptor expressed by T lymphocytes, has emerged as an effective therapy for the treatment of metastatic melanoma. Although subject to debate, current models favor a mechanism of activity involving blockade of the inhibitory activity of CTLA-4 on both effector (T eff) and regulatory (T reg) T cells, resulting in enhanced antitumor effector T cell activity capable of inducing tumor regression. We demonstrate, however, that the activity of anti–CTLA-4 antibody on the T reg cell compartment is mediated via selective depletion of T reg cells within tumor lesions. Importantly, T reg cell depletion is dependent on the presence of Fcγ receptor–expressing macrophages within the tumor microenvironment, indicating that T reg cells are depleted in trans in a context-dependent manner. Our results reveal further mechanistic insight into the activity of anti-CTLA-4–based cancer immunotherapy, and illustrate the importance of specific features of the local tumor environment on the final outcome of antibody-based immunomodulatory therapies.

BACKGROUND In this study, we investigated the effects of combining lenalidomide and docetaxel on in vitro and in vivo models of prostate cancer as a potential strategy for treatment of castrate resistant prostate cancer (CRPC). METHODS The effects of combining lenalidomide and docetaxel on proliferation, apoptosis, invasive potential, anchorage independent growth, and p53 activation in the PC3 and DU145 prostate cell lines were investigated. The effects of the lenalidomide and docetaxel combination on LNCaP prostate cancer cell growth and invasiveness in vitro was also studied. The combination of these two agents was finally tested on a xenograft model of PC3 tumor growth in nude mice. RESULTS Lenalidomide decreased the IC50 of docetaxel by up to 50% (P

Targeting CD38 in multiple myeloma has resulted in outstanding responses. CD38 is widely expressed on myeloma cells and other hematological malignancies. Not much is known about its expression on solid tumors and its role in the immune system. We have analysed a range of solid tumors for CD38 expression and distribution. To optimally target CD38, we have generated a novel antibody that is depleting CD38-high expressing cells, but also has immune modulatory properties. To dissect CD38 expression in solid tumors we exploited mRNA expression libraries, performed immune histochemistry (IHC) on tumor sections, and flow cytometry on patient tumor material. Bioinformatic analysis of the immune cell atlas revealed varying CD38 expression among all cancers analysed, and CD38 expression could be correlated with immune markers, e.g. Foxp3, PD-1/L1. IHC and flow cytometry confirmed CD38 expression across common cancer types, mostly confined to infiltrating lymphocyte and myeloid subsets. Expression on tumor cells was patient dependent. CD38 expression on immune cells was heterogenous and found on NK cells, T cells, suppressive myeloid cells, as well as regulatory T and B cells. Of note, high expression of CD38 was found to be correlated to a subset of exhausted T cells co-expressing PD-1 and other exhaustion markers. To target CD38 in solid tumors, we have screened a panel of CD38-binding antibodies. All antibodies have the potential to deplete CD38 positive tumor cells in vitro and in vivo. Additionally, their ability to influence effector T and NK cell activation has been evaluated. Among a panel of antibodies binding to distinct epitopes of CD38 and exerting unique functional properties, we have identified a fully human antibody, with strong capacity to deplete CD38-high cells in vitro and in vivo by varying killing mechanisms. This antibody was found to increase TCR-mediated signaling and proinflammatory cytokine secretion by human T cells, and further to enhance NK cell activation in vitro. Low dose injection to non-human primates resulted in increased expression of activation markers on both CD4 and CD8 T cells, while no T cell depletion was observed. Other selected antibodies comprise distinct modalities including strong to weak agonistic activity, differential killing properties, modulation of CD38 enzymatic activity, and offer a selection of candidates applicable for different treatment settings. In summary, we found heterogenous expression of CD38 in solid tumors, mostly confined to immune subsets. To target CD38, we present a potent anti-CD38 antibody with depleting effects on CD38-expressing cancer cells, as well as suppressive immune cells, and the capacity to increase the function of immune effector cells. This dual activity might allow to fully exploit the therapeutic potential of targeting CD38, not only in hematological malignancies but also in solid tumors. Citation Format: Nina Eissler, Simone Filosto, Jake Y. Henry, Michael F. Maguire, Kristina Witt, Andreas Lundqvist, Teresa Marafioti, Pascal Merchiers, Kevin Moulder, Beatriz Goyenechea, Haw Lu, Camilla Fairbairn, Sarah Windler, JD Aurellano, Omar Duramad, Dominic Smethurst, Sergio A. Quezada, Anne Goubier. A best in class anti-CD38 antibody with antitumor and immune-modulatory properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3812.

BACKGROUND AND PURPOSE We have investigated the distribution of α-adrenoceptors in sheep internal anal sphincter (IAS), as a model for the human tissue, and evaluated various imidazoline derivatives for potential treatment of faecal incontinence. EXPERIMENTAL APPROACH Saturation and competition binding with 3H-prazosin and 3H-RX821002 were used to confirm the presence and density of α-adrenoceptors in sheep IAS, and the affinity of imidazoline compounds at these receptors. A combination of in vitro receptor autoradiography and immunohistochemistry was used to investigate the regional distribution of binding sites. Contractile activity of imidazoline-based compounds on sheep IAS was assessed by isometric tension recording. KEY RESULTS Saturation binding confirmed the presence of both α1- and α2-adrenoceptors, and subsequent characterization with sub-type-selective agents, identified them as α1A- and α2D-adrenoceptor sub-types. Autoradiographic studies with 3H-prazosin showed a positive association of α1-adrenoceptors with immunohistochemically identified smooth muscle fibres. Anti-α1-adrenoceptor immunohistochemistry revealed similar distributions of the receptor in sheep and human IAS. The imidazoline compounds caused concentration-dependent contractions of the anal sphincter, but the maximum responses were less than those elicited by l-erythro-methoxamine, a standard non-imidazoline α1-adrenoceptor agonist. Prazosin (selective α1-adrenoceptor antagonist) significantly reduced the magnitude of contraction to l-erythro-methoxamine at the highest concentration used. Both prazosin and RX811059 (a selective α2-adrenoceptor antagonist) reduced the potency (pEC50) of clonidine. CONCLUSIONS AND IMPLICATIONS This study shows that both α1- and α2-adrenoceptors are expressed in the sheep IAS, and contribute (perhaps synergistically) to contractions elicited by various imidazoline derivatives. These agents may prove useful in the treatment of faecal incontinence.

The Drosophila head vertex is composed of three ocelli, stereotypic bristle patterns and characteristic cuticles. It is derived from the fusion of two eye-antenna discs. The head vertex primordium is located at the anterior-dorsal region of the eye disc. The orthodenticle (otd) homeobox gene is expressed in the primordium and is functionally required for its development and patterning. Here we show that the Pax gene eye gone (eyg) is expressed adjacent to the otd expression domain in the eye disc. otd is required and sufficient to repress eyg transcription, thereby preventing eyg from expressing in the head vertex primordium. In otd mutant, eyg expression is derepressed in the head vertex primordium and is a major negative effector to block head vertex development. Therefore, otd not only needs to induce downstream effector genes to execute the development and patterning of the head vertex development, but also needs to actively repress the negative regulator eyg. In addition, eyg is required for the development of the lateral bristles in the head vertex. So eyg plays both positive and negative roles in head vertex development.

Many illnesses are associated with an alteration of the immune system homeostasis due to a combination of factors, including exogenous bacterial insult, endogenous breakdown (e.g. development of a disease that results in immuno suppression), or an exogenous hit like surgery that simultaneously alters immune responsiveness and provides access to bacteria, or genetic disorder. We conjecture that, as a consequence of the co-evolution of the human immune system with the ecology of pathogens, the homeostasis of the immune system requires the influx of pathogens. This allows the immune system to keep the ever-present pathogens under control and to react and adjust fast to bursts of infections. We construct the simplest and most general system of rate equations which describes the dynamics of five compartments: healthy cells, altered cells, adaptive immune cells, innate immune cells, and pathogens. We study four regimes obtained with or without auto-immune disorder and with or without spontaneous proliferation of infected cells. For each of the four regimes, the phase space is always characterized by four (but not necessary identical) coexisting stationary structurally stable states. Over all four regimes among the 4 × 4 possibilities, there are only seven different states that are naturally described by the model: (i) strong healthy immune system, (ii) healthy organism with evanescent immune cells, (iii) chronic infections, (iv) strong infections, (v) cancer, (vi) critically ill state, and (vii) death. Our description provides a natural framework for describing the relationships and transitions between these seven states. The analysis of stability conditions demonstrates that these seven states depend on the balance between the robustness of the immune system and the influx of pathogens. In particular, the healthy state A is found to exist only under the influence of a sufficiently large pathogen flux, which suggests that health is not the absence of pathogens, but rather a strong ability to find balance by counteracting any pathogen attack.

Lenalidomide (Revlimid; CC-5013) and pomalidomide (CC-4047) are IMiDs proprietary drugs having immunomodulatory properties that have both shown activity in cancer clinical trials; lenalidomide is approved in the United States for a subset of MDS patients and for treatment of patients with multiple myeloma when used in combination with dexamethasone. These drugs exhibit a range of interesting clinical properties, including anti-angiogenic, anti-proliferative, and pro-erythropoietic activities although exact cellular target(s) remain unclear. Also, anti-inflammatory effects on LPS-stimulated monocytes (TNF-alpha is decreased) and costimulatory effects on anti-CD3 stimulated T cells, (enhanced T cell proliferation and proinflammatory cytokine production) are observed. These drugs also cause augmentation of NK-cell cytotoxic activity against tumour-cell targets. Having shown that pomalidomide confers T cell-dependent adjuvant-like protection in a preclinical whole tumour-cell vaccine-model, we now show that lenalidomide and pomalidomide strongly inhibit T-regulatory cell proliferation and suppressor-function. Both drugs inhibit IL-2-mediated generation of FOXP3 positive CTLA-4 positive CD25high CD4+ T regulatory cells from PBMCs by upto 50%. Furthermore, suppressor function of pre-treated T regulatory cells against autologous responder-cells is abolished or markedly inhibited without drug related cytotoxicity. Also, Balb/C mice exhibit 25% reduction of lymph-node T regulatory cells after pomalidomide treatment. Inhibition of T regulatory cell function was not due to changes in TGF-beta or IL-10 production but was associated with decreased T regulatory cell FOXP3 expression. In conclusion, our data provide one explanation for adjuvant properties of lenalidomide and pomalidomide and suggest that they may help overcome an important barrier to tumour-specific immunity in cancer patients.

The intent of this study was to ascertain to what extent serum total calcium concentration (tCa)7 mg/dL reflects hypocalcemia (defined by ionized calcium concentration [iCa] ofor = 1.12 mmol/L) in critically ill patients receiving specialized nutrition support.Adult patients (or = 18 years) admitted to the trauma, surgical, medical, burn, or neurosurgical intensive care units, trauma stepdown unit, or progressive care unit and referred to the nutrition support service were retrospectively identified for potential inclusion into the study. Serum chemistries, arterial blood gas measurements, nutrition markers, and serum iCa were simultaneously obtained from each patient approximately 1 day after initiation of specialized nutrition support. Patients with a serum creatinineor = 2 mg/dL, hyperphosphatemia (or = 6 mg/dL), severe hypomagnesemia (or = 1.12 mg/dL), history of metabolic bone disease, or parathyroid disease were excluded from the analysis.One hundred ninety-five patients (91% who had multiple trauma, with a mean Injury Severity Score 31 +/- 13) were enrolled into the study. Specialized nutrition support was initiated 2.8 +/- 1.8 days and calcium status was studied 4.2 +/- 3.1 days after hospital admission, respectively. The majority (28 of 33, or 85%) of patients with a tCa7 mg/dL were hypocalcemic compared with 33% (22 out of 66) of patients with a tCa of 7-7.4 mg/dL, and 11% (11 of 96) of those with a tCa of 7.5-7.9 mg/dL (p.001).Critically ill patients with a serum total calcium concentration of7 mg/dL have a high rate of hypocalcemia (iCaor = 1.12 mmol/L). Hypocalcemia, defined as a serum iCa ofor = 1.12 mmol/L, occurs in 85% of acutely ill patients with a serum tCa7 mg/dL.

As tumors grow, they acquire mutations, some of which create neoantigens that influence the response of patients to immune checkpoint inhibitors. We explored the impact of neoantigen intratumor heterogeneity (ITH) on antitumor immunity. Through integrated analysis of ITH and neoantigen burden, we demonstrate a relationship between clonal neoantigen burden and overall survival in primary lung adenocarcinomas. CD8(+)tumor-infiltrating lymphocytes reactive to clonal neoantigens were identified in early-stage non-small cell lung cancer and expressed high levels of PD-1. Sensitivity to PD-1 and CTLA-4 blockade in patients with advanced NSCLC and melanoma was enhanced in tumors enriched for clonal neoantigens. T cells recognizing clonal neoantigens were detectable in patients with durable clinical benefit. Cytotoxic chemotherapy-induced subclonal neoantigens, contributing to an increased mutational load, were enriched in certain poor responders. These data suggest that neoantigen heterogeneity may influence immune surveillance and support therapeutic developments targeting clonal neoantigens.

Cytomegalovirus (CMV) infection is responsible for substantial morbidity and mortality after allogeneic hematopoietic stem cell transplant. T-cell immunity is critical for control of CMV infection, and correction of the immune deficiency induced by transplant is now clinically achievable by the adoptive transfer of donor-derived CMV-specific T cells. It is notable, however, that most clinical studies of adoptive T- cell therapy exclude patients with graft-versus-host disease (GVHD) from receiving systemic corticosteroid therapy, which impairs cellular immunity. This group of patients remains the highest clinical risk group for recurrent and problematic infections. Here, we address this unmet clinical need by genetic disruption of the glucocorticoid receptor (GR) gene using electroporation of transcription activator-like effector nuclease (TALEN) messenger RNA. We demonstrate efficient inactivation of the GR gene without off-target activity in Streptamer-selected CMV-specific CD8(+) T cells (HLA-A02/NLV peptide), conferring resistance to glucocorticoids. TALEN-modified CMV-specific T cells retained specific killing of target cells pulsed with the CMV peptide NLV in the presence of dexamethasone (DEX). Inactivation of the GR gene also conferred resistance to DEX in a xenogeneic GVHD model in sublethally irradiated NOD-scid IL2rγ(null) mice. This proof of concept provides the rationale for the development of clinical protocols for producing and administering high-purity genetically engineered virus-specific T cells that are resistant to the suppressive effects of corticosteroids.

Organ formation requires a delicate balance of positive and negative regulators. In Drosophila eye development, wingless (wg) is expressed at the lateral margins of the eye disc and serves to block retinal development. The T-box gene optomotor-blind (omb) is expressed in a similar pattern and is regulated by Wg. Omb mediates part of Wg activity in blocking eye development. Omb exerts its function primarily by blocking cell proliferation. These effects occur predominantly in the ventral margin. Our results suggest that the primary effect of Omb is the blocking of Jak/STAT signaling by repressing transcription of upd which encodes the Jak receptor ligand Unpaired.

Cytomegalovirus (CMV) remains a significant cause of morbidity after allogeneic hematopoietic stem cell transplantation (HSCT). Clinical risk varies according to a number of factors, including recipient/donor CMV serostatus. Current dogma suggests risk is greatest in seropositive recipient (R+)/seronegative donor (D-) transplants and is exacerbated by T-cell depletion. We hypothesized that in the setting of reduced-intensity T-cell-depleted conditioning, recipient-derived CMV-specific T cells escaping deletion may contribute significantly to CMV-specific immunity and might therefore also influence chimerism status. We evaluated 105 recipients of alemtuzumab-based reduced-intensity HSCT and collated details on CMV infection episodes and T-cell chimerism. We used CMV-specific HLA multimers to enumerate CMV-specific T-cell numbers and select cells to assess chimerism status in a subset of R+/D- and R+/seropositive donor patients. We show that in R+/D- patients, CMV-specific T cells are exclusively of recipient origin, can protect against recurrent CMV infections, and significantly influence the chimerism status toward recipients. The major findings were replicated in a separate validation cohort. T-cell depletion in the R+/D- setting may actually, therefore, foster more rapid reconstitution of protective antiviral immunity by reducing graft-vs-host directed alloreactivity and the associated elimination of the recipient T-cell compartment. Finally, conversion to donor chimerism after donor lymphocytes is associated with clinically occult transition to donor-derived immunity.

The late onset of neurodegeneration in humans indicates that the survival and function of cells in the nervous system must be maintained throughout adulthood. In the optic lamina of the adult Drosophila, the photoreceptor axons are surrounded by multiple types of glia. We demonstrated that the adult photoreceptors actively contribute to glia maintenance in their target field within the optic lamina. This effect is dependent on the epidermal growth factor receptor (EGFR) ligands produced by the R1-6 photoreceptors and transported to the optic lamina to act on EGFR in the lamina glia. EGFR signaling is necessary and sufficient to act in a cell-autonomous manner in the lamina glia. Our results suggest that EGFR signaling is required for the trafficking of the autophagosome/endosome to the lysosome. The loss of EGFR signaling results in cell degeneration most likely because of the accumulation of autophagosomes. Our findings provide in vivo evidence for the role of adult neurons in the maintenance of glia and a novel role for EGFR signaling in the autophagic flux., Author Summary Degeneration of the nervous system can be viewed as a failure to maintain cell survival or function in the nervous system. The late onset of neurodegeneration in humans indicates that the cell survival in the nervous system must be maintained throughout our lives. Neuronal survival is maintained by neurotrophic factors in adults; however, it is unclear whether glia survival is also maintained throughout adulthood. Here, we use the Drosophila visual system as a model to address the role played by adult neurons for the active maintenance of glia. We demonstrated that the adult photoreceptors secrete a signaling molecule, which is transported to the brain to act on the lamina glia and maintain its integrity. When this signaling pathway is blocked, the lamina glia undergoes a progressive and irreversible degeneration. The primary defect occurs in the trafficking from the late endosome and autophagosome to the lysosome. This defect leads to an accumulation of autophagosomes and subsequent cell degeneration as a result of autophagy. Our findings provide in vivo evidence for a novel aspect of the neuron-glia interaction and a novel role for EGFR signaling in regulating the maintenance and degeneration of the nervous system.

Prostate cancer is the leading cause of cancer death among older men in western countries. However, controversy surrounds many issues related to this disease, particularly its most appropriate treatment, with a wide spectrum of opinions ranging from watchful waiting to aggressive therapy. Patients with newly diagnosed prostate cancer, as well as their doctors, will have to make difficult decisions regarding treatment of this disease. In this article we discuss the current available treatment options and some novel therapeutic approaches to tackling the patient with prostate cancer.

We characterized the changes in nitric oxide (NO) levels in the brain during global forebrain ischemia and reperfusion and tested the ability of the natural flavonoid, quercetin, and a synthetic flavonoid, FB277, to increase the amount of available NO by elimination of the superoxide radicals produced during reperfusion. In Sprague-Dawley rats, we used a four-vessel occlusion model of forebrain ischemia (15 min) and reperfusion (30 min). Brain NO was measured on samples of cerebral cortex and cerebellum ex vivo by electron paramagnetic resonance (EPR) spectroscopy. The spin trap used was diethyldithiocarbamate sodium salt (DETC) associated with ferrous citrate. The complex Fe(DETC)2NO was detected at 77 K as a triplet signal at g = 2.035. Groups of animals were treated with quercetin or FB277 (3-morpholinomethyl-3',4',5,7tetramethoxyflavone) or polyethylene glycol-conjugated superoxide dismutase (PEG-SOD). In control (intact anesthetized animals), the signal was about 3 times greater in the cortex than in the cerebellum. During ischemia, the signal rose to 110% in cortex (NS) and 283% in cerebellum (P < 0.05). In reperfusion, it fell again to 91% of control in cerebellum (NS) and 35% in cortex (P < 0.05). Treatment by quercetin (5 mg/kg i.v.) of intact and ischemia-reperfusion groups did not significantly change the signal amplitude in the cerebellum, but did double it in the cortex (to 76% of control) for the ischemia-reperfusion group (P < 0.05). In contrast, FB277 (3.75 mg/kg i.v.) did not increase the signal in the cortex during ischemia-reperfusion, but did do so in the cerebellum (to 152% of control, P < 0.05). The results obtained for PEG-SOD (10,000 U/kg i.v.) were similar to those for FB277. In separate in vitro measurements, we found that quercetin but not FB277 efficiently scavenged superoxide. We hypothesize that quercetin but not FB277 scavenged superoxide anions released in the cortex during reperfusion, thus diminishing the amount of NO removed by the formation of peroxynitrite. The lack of effect of PEG-SOD may be related to the need for chronic treatment to obtain protection.

Background The role of α-adrenoceptors in promoting continence through modulation of sphincter tone has focused primarily on the effects of α1-adrenoceptors. We have used three clinically available agents, which are selective for α2-adrenoceptors, to investigate their role in contractile and neurogenic responses on the internal anal sphincter (IAS). Methods IAS strips, which had spontaneously generated tone, were used to investigate the contractile effect of lofexidine, brimonidine, and dexmedetomidine on muscle tone in the presence or absence of subtype selective antagonists. The effect of brimonidine on the magnitude and time course of neurogenic responses generated by electrical field stimulation (EFS) was also examined. The affinity of test compounds at α1- and α2-adrenoceptors was established by competition binding with [3H]-prazosin and [3H]-RX821002. Key Results All agonists caused concentration-dependent contraction of the IAS and lofexidine demonstrated an enantiomeric difference in potency with a 10-fold difference between the (−) and (+) isomers. Responses to lofexidine and dexmedetomidine were inhibited in the presence of the α1-adrenoceptor selective antagonist prazosin, but not in the presence of RX811059 (α2-adrenoceptor selective antagonist); brimonidine responses were inhibited by RX811059 and, to a lesser extent, by prazosin. Brimonidine affected both magnitude and duration of neurogenic responses, which was reversed in the presence of RX811059. Conclusions & Inferences We conclude that α2-adrenoceptors can mediate contraction of IAS, although this effect is most evident with efficacious imidazoline agonists rather than the most selective ligand. In addition, this receptor subtype can directly inhibit noradrenergic contractile responses to EFS and, indirectly, enhance nitrergic relaxatory responses.

Bacterial formation of carcinogenic N-nitroso compounds may play a role in the etiology of human cancer. Biochemical and immunological studies in denitrifying bacteria (Pseudomonas aeruginosa) strongly support the identification of cytochrome cd(1)-nitrite reductase as the enzyme responsible for the catalysis of nitrosation through the production of nitric oxide or NO(+)-like species. Interestingly, electron paramagnetic resonance studies have shown that large quantities of nitric oxide or NO(+)-species were also produced by non-denitrifying enterobacteria (Escherichia coli, Proteus morganii).

The IMiDs(®) immunomodulatory compounds lenalidomide and pomalidomide are agents with anti-inflammatory, immunomodulatory and anti-cancer activity. An excellent success rate has been shown for multiple myeloma in phase I/II clinical trials leading to Food and Drug Administration approval of lenalidomide. One mechanism by which these drugs could enhance anti-tumour immunity may be through enhanced dendritic cell (DC) function. Thalidomide, a compound structurally related to lenalidomide and pomalidomide, is known to enhance DC function, and we have investigated whether its analogues, pomalidomide and lenalidomide, also have functional effects on DCs. We used mouse bone marrow-derived DCs treated with 5 or 10 μm pomalidomide, or lenalidomide from day 1 of culture. Treatment with IMiD(®) immunomodulatory compounds increased expression of Class I (H2-Kb), CD86, and pomalidomide also increased Class II (I-Ab) expression in bone marrow-derived DCs, as measured by flow cytometry. Fluorescent bead uptake was increased by up to 45% when DCs were treated with 5 or 10 μm pomalidomide or lenalidomide compared with non-treated DCs. Antigen presentation assays using DCs primed with ovalbumin, and syngeneic T cells from transgenic OTI and OTII mice (containing MHC restricted, ovalbumin-specific, T cells) showed that both pomalidomide and lenalidomide effectively increased CD8(+) T-cell cross-priming (by up to 47%) and that pomalidomide alone was effective in increasing CD4(+) T-cell priming (by 30%). Our observations suggest that pomalidomide and lenalidomide enhance tumour antigen uptake by DCs with an increased efficacy of antigen presentation, indicating a possible use of these drugs in DC vaccine therapies.

Nitric oxide is synthesized in mammalian cells from L-arginine or from pharmaceutical drugs. It forms paramagnetic complexes with some metalloproteins, inhibiting key enzymes in DNA synthesis, mitochondrial respiration, iron metabolism, etc. This article reviews how electron paramagnetic resonance spectroscopy helps to detect unambiguously such specific molecular targets for NO in mammalian whole cells and organelles. EPR has also been used for the detection of spin adducts of free NO by spin-trapping methods.

International audience; Rat liver microsomes catalyze the oxidative denitration of N omega-hydroxy-L-arginine (NOHA) by NADPH and O2 with formation of citrulline and nitrogen oxides like NO and NO2-. Besides NO2- and citrulline, whose simultaneous formation is linear for at least 20 min, the formation of NO could be detected under the form of its P450 and P420-Fe(II) complexes by UV-visible and EPR spectroscopy. Classical inhibitors of NO-synthases, like N omega-methyl-and N omega-nitro-arginine, fail to inhibit the microsomal oxidation of NOHA to citrulline and NO2-. On the contrary classical inhibitors of hepatic cytochromes P450 like CO, miconazole, dihydroergotamine and troleandomycin, strongly inhibit this monooxygenase reaction. These results show that the oxygenation of NOHA by NADPH and O2 with formation of citrulline and NO can be efficiently catalyzed by cytochromes P450 (with rates up to 1.5 turnovers per min for the cytochromes of the 3A subfamily).Rat liver microsomes catalyze the oxidative denitration of N omega-hydroxy-L-arginine (NOHA) by NADPH and O2 with formation of citrulline and nitrogen oxides like NO and NO2-. Besides NO2- and citrulline, whose simultaneous formation is linear for at least 20 min, the formation of NO could be detected under the form of its P450 and P420-Fe(II) complexes by UV-visible and EPR spectroscopy. Classical inhibitors of NO-synthases, like N omega-methyl-and N omega-nitro-arginine, fail to inhibit the microsomal oxidation of NOHA to citrulline and NO2-. On the contrary classical inhibitors of hepatic cytochromes P450 like CO, miconazole, dihydroergotamine and troleandomycin, strongly inhibit this monooxygenase reaction. These results show that the oxygenation of NOHA by NADPH and O2 with formation of citrulline and NO can be efficiently catalyzed by cytochromes P450 (with rates up to 1.5 turnovers per min for the cytochromes of the 3A subfamily).

The effects of dietary level of tryptophan (TRP) and CP content and composition on voluntary feed intake, growth performance, and carcass characteristics in finishing pigs were studied in two experiments, with an equal number of females and castrated males. In Exp. 1, involving 120 Large White pigs from 44 to 99 kg BW with ad libitum access to feed, six treatments were compared according to a 2 x 3 factorial arrangement: 1) two levels of TRP (.09 and .13%), suboptimal and optimal for growth, respectively, 2) three types of CP supply (a 12.5% CP diet based on corn-soybean meal, and adequately balanced for essential amino acids [EAA] other than TRP; 15.7% CP diet with additional protein from corn gluten meal; 16.2% CP diet with additional nonessential amino acids [NEAA, in the form of L-glutamic acid.HCl and glycine], and the same levels of EAA as in the 12.5% CP diet. In Exp. 2, including four of the six previous factorial combinations (.09 and .13% TRP, 12.3 and 15.8% CP with additional protein), 32 pigs of 50-kg initial BW were used during 21 d, and further observations on meat quality characteristics, plasma free amino acid levels, and serotonin concentrations in the posterior hypothalamus were made. The major observed effects were interactions of different magnitude according to sex between TRP level and the amount and the composition of additional CP. At the suboptimal level of .09% TRP, the increase in protein content severely decreased daily feed intake and growth compared with the .13% level, especially in females. Conversely, the addition of NEAA at both TRP levels had little effect on daily feed intake and growth. Deficiency of TRP exerted a significant increase of pH in adductor femoris and semimembranosus muscles measured 45 min and 24 h postmortem, but only in females. Voluntary feed intake, as affected by dietary TRP and CP levels, was linearly related with concomitant changes in TRP to large neutral amino acids (TRP:LNAA) ratio both in feed and in plasma, which in turn was directly associated to hypothalamic serotonin concentration. It was concluded that an overly low concentration of serotonin in the hypothalamus, especially in females, as a result of TRP:LNAA imbalance, could be involved in the reduction of voluntary feed intake.

The effects of dietary level of lysine and of level and source of CP on voluntary feed intake, growth performance, plasma free amino acids, and carcass characteristics were investigated in a study involving 60 female and 60 castrated male Large White finishing pigs (from 42 to 101 kg live weight) with ad libitum access to feed. Six treatments were compared according to a 2 x 3 factorial plan, with two levels of lysine (.55 and .65% selected below the recommended levels for both sexes) and three types of CP (N x 6.25) supply: a 13% CP diet based on wheat, peanut meal and soybean meal; a 15.6% CP diet providing the same amino acid pattern as that of the basal diet; and a 15.2% CP diet containing the same levels of essential amino acids as the 13% CP diet, with the addition of glutamic acid as a source of nonessential amino acids. By maintaining a constant amino acid pattern separate changes in dietary lysine and CP levels resulted in a relative independency of their effects on feed intake, growth performance, and body composition. Muscle gain increased with supplementary lysine, with a lower response at the lower CP level (13%). At the same level of lysine (.55 or .65%), increasing protein content from 13 to 15.6% did not affect feed intake, but growth rate was lower and feed/gain was increased, partly because of an additional energy cost resulting from catabolism of excess protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Background: Thalidomide and lenalidomide are FDA approved for the treatment of multiple myeloma and, along with pomalidomide, are being investigated in various other cancers. Although these agents display immunomodulatory, anti-angiogenic and anti-apoptotic effects, little is known about their primary mode of therapeutic action in patients with cancer. Methods: As part of a continuing research effort, we have investigated the effects of these agents on the metastatic capacity of murine colorectal cancer cell lines both in vivo and in vitro. Allied to these, we have studied their effects on the molecular pathways associated with metastasis. Results: Results indicate that thalidomide, lenalidomide and pomalidomide significantly inhibit the metastatic capability of colorectal carcinoma cells. Anchorage-independent growth, used as a coarse indicator of transformation, was significantly reduced, as were migratory capacity and invasive competence. In addition, an in vivo experimental metastasis model also showed that treatment with the drugs resulted in a significantly lower number of metastatic pulmonary nodules relative to control mice. Allied to these cellular and phenotypic changes were alterations in molecular markers of metastasis and in intracellular signalling competency. Conclusions: These results provide evidence that in addition to their immunomodulatory effects, thalidomide, lenalidomide and pomalidomide can impair the metastatic capacity of tumours, and that this mechanism may involve alterations to cell signalling functionality.

The behavioral reactivity in an "open-field" test and plasma cortisol levels were studied in 72 pigs from 12 litters fed for 3 wk one of three diets with different levels of tryptophan: deficient (.14%), adequate (.23%), or excess (.32%). "Open-field" tests were performed three times: 5 d (day W + 5), 23 d (day W + 23) and 45 d (day W + 45) after weaning. The exploration time and the number of grunts provided an adequate measure of the individual emotional reactivity at day W + 5. Significant correlations were obtained between exploration time and the number of grunts at each time (r = -.83 at day W + 5; r = -.46 at day W + 23; r = -.71 at day W + 45). The distinction between animals remained (P less than .05) in terms of exploration time at both 23 and 45 d after weaning. At day W + 23, exploration time was lower in the group fed the adequate diet than in the two other groups. This effect was maintained subsequently after feeding all pigs the same adequate diet (day W + 45). In 36 pigs slaughtered at day W + 23, brain TRP concentration was higher with the excess dietary TRP than with deficient or adequate levels. Conversely, other plasma amino acids (particularly threonine) accumulated only in the brains of pigs fed the deficient diet. Plasma cortisol level assayed at weaning (W) and 2 wk later increased with age and was higher in 16-h fasted (day W + 15) than in 3-h fasted (day W + 17) pigs. Correlations were observed within litters in the fasting state, between the cortisol level and behavioral traits measured at day W + 23 (r = .70 for number of grunts, r = -.60 for exploration time). Dietary TRP did not affect the plasma cortisol level irrespective of the nutritional state after weaning. However, an interaction was noted between plasma cortisol and TRP status (P less than .05). Although dietary TRP induced large variations in brain amino acids and 5-hydroxyindole concentrations, changes in behavioral and cortisol responses were relatively minor.

After immunostimulation, murine macrophages oxidize L-arginine into nitric oxide (NO) which acts as an effector molecule. In this study, we attempted to establish whether activated macrophage-derived NO forms paramagnetic complexes in tumor target cells which do not express by themselves the L-arginine:NO pathway. Accordingly, murine L1210 leukemia cells were cocultivated with activated peritoneal macrophages from Bacillus-Calmette-Guerin-infected mice, or activated in vitro with interferon-gamma. In control experiments, macrophages were prevented from producing nitrogen oxides by incubation with NG-monomethyl-L-arginine, a specific inhibitor of the L-arginine:NO pathway. After coculture, L1210 cells were removed from adherent macrophage monolayers and analyzed by electron paramagnetic resonance at 77 K. In the L1210 cells cultured with activated macrophages, we detected a signal typical of nitrosyl-iron-sulfur complexes, with g values of 2.041 and 2.015. This signal was not present when L1210 cells were either cultured alone or cocultured with activated macrophages in the presence of NG-monomethyl-L-arginine. Mitochondria from activated macrophage-injured L1210 cells also exhibited the signal with g values of 2.041 and 2.015. These results show that when tumor target cells undergo cell-to-cell contact with activated macrophages during culture, the macrophages promote target cell nitrosylation in compartments like mitochondria.

Malignant tumors maintain a dominant immunosuppressive microenvironment that facilitates their progression and dissemination, mediated by the secretion of soluble factors, recruitment of different subsets of immune cells and expression of immune-regulatory receptors. The immune-modulatory cytokine interleukin-12 (IL-12) is a promising alternative to overcome this suppressive microenvironment because of its direct role in the activation of innate and adaptive immunity and potent anti-angiogenic effect. Despite its potent activity in experimental models, the clinical use of IL-12 for the treatment of human cancers has proven difficult because of its toxicity. The lung is a preferred site of metastasis for several types of tumor. Therefore, we hypothesized that local delivery of IL-12 could improve the immune response against lung metastases via modification of the tumor microenvironment and avoiding systemic toxicity. Therefore, we evaluated the anti-tumor activity of IL-12 in a murine model of metastatic B16 melanoma by intranasal delivery lentiviral vector encoding IL-12 (IL-12 LV). This delivery route resulted in transduction of alveolar macrophages and a drastic reduction of lung metastases. Whilst treated mice survived longer than controls, they eventually succumbed to distant tumor lesions while their lungs remained tumor-free. IL-12 LV treatment was associated with local infiltration and proliferation of effector subsets (NK, CD8+, CD4+ T cells) that expressed high levels of granzyme B and pro-inflammatory cytokines. These cells, however, are dispensable as IL12 LV was equally effective in models of NK and T cell ablation. Interferon gamma (IFNγ) was identified as an essential cytokine in the mechanism of IL-12 LV mediated tumoricidal activity. IFNg expression under IL-12 LV treatment was observed in NK, T cell and Innate Like Cell (ILC) populations, the latter of which sufficiently sustains IFNγ dependant activity in the absence of NK and T cells. The myeloid populations of the IL-12 LV treated lung were phenotyped and expansion of Interstitial Macrophages with high expression of MHC class II and inducible nitric oxide synthase (iNOS) was observed in an IFNγ dependant manner, therefore transgenic models of macrophage associated IFNγ insensitivity are currently being employed in order to elucidate the essential cytotoxic status of Interstitial Macrophages in models of IL12-LV mediated tumor rejection. We conclude that activation of innate and adaptive immunity by local genetic delivery of IL-12 is effective for the treatment of metastatic melanoma in the lung. Despite its local effectiveness, systemic protection might require further combination therapy, such as vaccination or checkpoint inhibition. Citation Format: Jake Y. Henry, Frederick Arce, Burkhard Becher, Karl S. Peggs, Sergio A. Quezada. Innate crosstalk between ILC and interstitial macrophages promotes lung cancer regression in response to IL-12 therapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A127.

The radiosensitizing effect of nitric oxide (NO) on mouse and human tumor cells with different degrees of malignancy was evaluated. Cells pre-treated with the NO donor diethylenetriamine-NO (DETA-NO), were irradiated with γ rays. Survival curves were obtained by clonogenicity and fitted to the linear–quadratic model. Results demonstrated an association between radiosensitization and degree of malignancy. The more malignant the cell line, the higher the degree of radiosensitization by DETA-NO. In conclusion, the differential radiosensitizing effect of DETA-NO shown here is of great interest for the potential use of NO in radiotherapy, due to an enhanced radiation effect on tumor vs. normal tissue.

Cyclooxygenase-2 (COX-2) action on the endocannabinoids, 2-arachidonylglycerol (2-AG) and anandamide (AEA), generates prostaglandin glycerol esters (PG-G) and ethanolamides (PG-EA), respectively. The diversity of PG-Gs and PG-EAs that can be formed enzymatically following COX-2 oxygenation of endocannabinoids was examined in cellular and subcellular systems. In cellular systems, glycerol esters and ethanolamides of PGE(2), PGD(2), and PGF(2alpha) were major products of the endocannabinoid-derived COX-2 products, PGH(2)-G and PGH(2)-EA. The sequential action of purified COX-2 and thromboxane synthase on AEA and 2-AG provided thromboxane A(2) ethanolamide and glycerol ester, respectively. Similarly, bovine prostacyclin synthase catalyzed the isomerization of the intermediate endoperoxides, PGH(2)-G and PGH(2)-EA, to the corresponding prostacyclin derivatives. Quantification of the efficiency of prostaglandin and thromboxane synthase-directed endoperoxide isomerization demonstrated that PGE, PGD, and PGI synthases catalyze the isomerization of PGH(2)-G at rates approaching those observed with PGH(2). In contrast, thromboxane synthase was far more efficient at catalyzing PGH(2) isomerization than at catalyzing the isomerization of PGH(2)-G. These results define the in vitro diversity of endocannabinoid-derived prostanoids and will permit focused investigations into their production and potential biological actions in vivo.