P75NTR+CD64+ neutrophils promote sepsis-induced acute lung injury.

Patients suffering from sepsis-induced acute lung injury (ALI) exhibit a high mortality rate, and their prognosis is closely associated with infiltration of neutrophils into the lungs. In this study, we found a significant elevation of CD64+ neutrophils, which highly expressed p75 neurotrophin receptor (p75NTR) in peripheral blood of mice and patients with sepsis-induced ALI. p75NTR+CD64+ neutrophils were also abundantly expressed in the lung of ALI mice induced by lipopolysaccharide. Conditional knock-out of the myeloid lineage's p75NTR gene improved the survival rates, attenuated lung tissue inflammation, reduced neutrophil infiltration and enhanced the phagocytic functions of CD64+ neutrophils. In vitro, p75NTR+CD64+ neutrophils exhibited an upregulation and compromised phagocytic activity in blood samples of ALI patients. Blocking p75NTR activity by soluble p75NTR extracellular domain peptide (p75ECD-Fc) boosted CD64+ neutrophils phagocytic activity and reduced inflammatory cytokine production via regulation of the NF-κB activity. The findings strongly indicate that p75NTR+CD64+ neutrophils are a novel pathogenic neutrophil subpopulation promoting sepsis-induced ALI.

Listening to the Whispers in Neuroimmune Crosstalk: A Comprehensive Workflow to Investigate Neurotrophin Receptor p75NTR Under Endogenous, Low Abundance Conditions.

Inflammatory conditions are critically influenced by neuroimmune crosstalk. Cytokines and neurotrophic factors shape the responses of both nervous and immune systems. Although much progress has been made, most findings to date are based on expression of recombinant (tagged) proteins. The examination of receptor interactions by immunoprecipitation (IP) at endogenous levels provides further insight into the more subtle regulations of immune responses. Here, we present a comprehensive workflow and an optimized IP protocol that provide step-by-step instructions to investigate neurotrophin receptor p75NTR at endogenous, low abundance levels: from lysate preparation and confirmation of receptor expression to antibody validation and successful detection of protein-protein interactions. We employ human melanoma cell line A375 to validate specific antibodies and IP conditions, and apply these methods to explore p75NTR interactions in human leukemic plasmacytoid dendritic cell line PMDC05 detecting 14-3-3ϵ:p75NTR interaction in this cell type. With p75NTR as an exemplary protein, our approach provides a strategy to detect specific interaction partners even under endogenous, low abundance expression conditions.

Evolution of delayed resistance to immunotherapy in a melanoma responder.

Despite initial responses1-3, most melanoma patients develop resistance4 to immune checkpoint blockade (ICB). To understand the evolution of resistance, we studied 37 tumor samples over 9 years from a patient with metastatic melanoma with complete clinical response to ICB followed by delayed recurrence and death. Phylogenetic analysis revealed co-evolution of seven lineages with multiple convergent, but independent resistance-associated alterations. All recurrent tumors emerged from a lineage characterized by loss of chromosome 15q, with post-treatment clones acquiring additional genomic driver events. Deconvolution of bulk RNA sequencing and highly multiplexed immunofluorescence (t-CyCIF) revealed differences in immune composition among different lineages. Imaging revealed a vasculogenic mimicry phenotype in NGFRhi tumor cells with high PD-L1 expression in close proximity to immune cells. Rapid autopsy demonstrated two distinct NGFR spatial patterns with high polarity and proximity to immune cells in subcutaneous tumors versus a diffuse spatial pattern in lung tumors, suggesting different roles of this neural-crest-like program in different tumor microenvironments. Broadly, this study establishes a high-resolution map of the evolutionary dynamics of resistance to ICB, characterizes a de-differentiated neural-crest tumor population in melanoma immunotherapy resistance and describes site-specific differences in tumor-immune interactions via longitudinal analysis of a patient with melanoma with an unusual clinical course.

Tissue-Specificity of Antibodies Raised Against TrkB and p75NTR Receptors; Implications for Platelets as Models of Neurodegenerative Diseases.

Platelets and neurons share many similarities including comparable secretory granule types with homologous calcium-dependent secretory mechanisms as well as internalization, sequestration and secretion of many neurotransmitters. Thus, platelets present a high potential to be used as peripheral biomarkers to reflect neuronal pathologies. The brain-derived neurotrophic factor (BDNF) acts as a neuronal growth factor involved in learning and memory through the binding of two receptors, the tropomyosin receptor kinase B (TrkB) and the 75 kDa pan-neurotrophic receptor (p75NTR). In addition to its expression in the central nervous system, BDNF is found in much greater quantities in blood circulation, where it is largely stored within platelets. Levels 100- to 1,000-fold those of neurons make platelets the most important peripheral reservoir of BDNF. This led us to hypothesize that platelets would express canonical BDNF receptors, i.e., TrkB and p75NTR, and that the receptors on platelets would bear significant resemblance to the ones found in the brain. However, herein we report discrepancies regarding detection of these receptors using antibody-based assays, with antibodies displaying important tissue-specificity. The currently available antibodies raised against TrkB and p75NTR should therefore be used with caution to study platelets as models for neurological disorders. Rigorous characterization of antibodies and bioassays appears critical to understand the interplay between platelet and neuronal biology of BDNF.

A novel immunocompetent model of metastatic prostate cancer-induced bone pain.

BACKGROUND: Over 70% to 85% of men with advanced prostate cancer (PCa) develop bone metastases characterized by severe bone pain and increased likelihood of bone fracture. These clinical features result in decreased quality of life and act as a predictor of higher mortality. Mechanistically, the skeletal pathologies such as osteolytic lesions and abnormal osteoblastic activity drive these symptoms. The role of immune cells in bone cancer pain remains understudied, here we sought to recapitulate this symptomology in a murine model. METHODS: The prostate cancer bone metastasis-induced pain model (CIBP) was established by transplanting a mouse prostate cancer cell line into the femur of immunocompetent mice. Pain development, gait dynamics, and the changes in emotional activities like depression and anxiety were evaluated. Animal tissues including femurs, dorsal root ganglion (DRG), and spinal cord were collected at killing and microcomputed tomography (µCT), histology/immunohistochemistry, and quantitative immunofluorescent analysis were performed. RESULTS: Mice receiving prostate cancer cells showed a significantly lower threshold for paw withdrawal responses induced by mechanical stimulation compared with their control counterparts. Zero maze and DigiGait analyses indicated reduced and aberrant movement associated emotional activity compared with sham control at 8-weeks postinjection. The µCT analysis showed osteolytic and osteoblastic changes and a 50% reduction of the trabecular volumes within the prostate cancer group. Neurologically we demonstrated, increased calcitonin gene-related peptide (CGRP) and neuronal p75NTR immune-reactivities in both the projected terminals of the superficial dorsal horn and partial afferent neurons in DRG at L2 to L4 level in tumor-bearing mice. Furthermore, our data show elevated nerve growth factor (NGF) and TrkA immunoreactivities in the same segment of the superficial dorsal horn that were, however, not colocalized with CGRP and p75NTR . CONCLUSIONS: This study describes a novel immunocompetent model of CIBP and demonstrates the contribution of NGF and p75NTR to chronic pain in bone metastasis.

Establishment of a Monoclonal Antibody That Recognizes Cysteine-Rich Domain 1 of Human CD271.

CD271 is a common receptor for all neurotrophins that is localized to neurons, endothelial cells, and the basal layer of the epithelium in normal tissue. Recently, we and others reported that CD271 plays essential roles in the development of squamous cell carcinoma, especially in tumor-initiating cells. Since little is known about how CD271 regulates cancer cell initiation and proliferation, antibodies that recognize different domains of CD271 are needed to enable investigation. Therefore, this study aimed to develop an antihuman CD271 antibody by immunizing mice with a CD271 antigen produced by a baculovirus. The antibody was named hCD271mAb#13, and it recognized cysteine-rich domain 1 with a higher affinity than the commercially available antibody ME20.4. We determined that hCD271mAb#13 is suitable for flow cytometry, Western blotting, immunocytochemistry, and immunohistochemistry of formalin-fixed paraffin-embedded tissue. Use of hCD271mAb#13 for CD271 labeling could enable detailed analyses of cancer cell regulation and other biological processes.

Anti-BCMA chimeric antigen receptors with fully human heavy-chain-only antigen recognition domains.

Chimeric antigen receptor (CAR)-expressing T cells targeting B-cell maturation antigen (BCMA) have activity against multiple myeloma, but improvements in anti-BCMA CARs are needed. We demonstrated recipient anti-CAR T-cell responses against a murine single-chain variable fragment (scFv) used clinically in anti-BCMA CARs. To bypass potential anti-CAR immunogenicity and to reduce CAR binding domain size, here we designed CARs with antigen-recognition domains consisting of only a fully human heavy-chain variable domain without a light-chain domain. A CAR designated FHVH33-CD8BBZ contains a fully human heavy-chain variable domain (FHVH) plus 4-1BB and CD3ζ domains. T cells expressing FHVH33-CD8BBZ exhibit similar cytokine release, degranulation, and mouse tumor eradication as a CAR that is identical except for substitution of a scFv for FHVH33. Inclusion of 4-1BB is critical for reducing activation-induced cell death and promoting survival of T cells expressing FHVH33-containing CARs. Our results indicate that heavy-chain-only anti-BCMA CARs are suitable for evaluation in a clinical trial.

Gene Modification and Immunological Analyses for the Development of Immunotherapy Utilizing T Cells Redirected with Antigen-Specific Receptors.

Cancer immunotherapy has been developed and established as a new treatment modality. Recently, adoptive transfer therapy using T cells redirected with antigen-specific antitumor receptors, such as T-cell receptor (TCR) and chimeric antigen receptor (CAR), has demonstrated clinical benefits even in patients with refractory malignancies. To advance this treatment modality, both generation of gene-modified T cells and evaluation of their reactivity with high quality in vitro are required. To achieve this, it is important to establish the ways (1) to generate optimal viral particle for T-cell transduction, (2) to transduce antitumor receptors into T cells and expand redirected T cells efficiently, and (3) to assess the functionality of antigen-specific gene-modified T cells precisely. Here, we summarize established protocols to generate and analyze antitumor receptor-transduced T cells. These procedures help to further assess characteristics of gene-modified T cells, resulting in promotion of translational research for cancer immunotherapy.

Humanized anti-CD271 monoclonal antibody exerts an anti-tumor effect by depleting cancer stem cells.

CD271, known as a neurotrophin receptor, is expressed in various cancers such as hypopharyngeal cancer (HPC) and melanoma. We recently reported that CD271 is a cancer-stem-cell biomarker of HPC, and that its expression is essential for cancer-cell proliferation and is correlated with a poor prognosis in this disease. Here, to develop a therapeutic antibody to CD271, we established a humanized anti-CD271 monoclonal antibody (hCD271 mA b). hCD271 mA b bound to the cysteine-rich domain 1 (CRD1) of human CD271 with high affinity (KD = 1.697 × 10-9 M). In vitro, hCD271 mA b exerted antibody-dependent cell-mediated cytotoxicity (ADCC) activity against SP2/0-CD271 (human CD271-transduced mouse cell line). Treatment with hCD271 mA b also exerted anti-tumor activity in graft models of three cell lines (HPCM2 (patient-derived xenograft cell line of hypopharyngeal cancer), MeWo-Luc (melanoma cell line), and SP2/0-CD271) in mice, resulting in smaller tumors compared to controls and reduced numbers of CD271-positive cells. Collectively, these data suggest that an antibody targeting CD271 is a promising therapeutic strategy.

Neurotrophic factors and nerve growth factor in ocular allergy.

PURPOSE OF REVIEW: During allergic reaction, nervous and immune systems mutually interact through release of mediators, including neurotrophic factors and nerve growth factor (NGF). These mediators modulate allergic reaction through binding their receptors expressed by immune and structural cells and by stimulating neuropeptide release by nerves. The role of neuropeptides and NGF has been demonstrated in allergic asthma and rhinitis, and, to a lesser extent, in allergic conjunctivitis. The aim of this review are to elucidate the evidence of the role of NGF and neuropeptides in the pathogenesis of allergic conjunctivitis. RECENT FINDINGS: NGF modulates allergic reaction by stimulating release of cytokines, inflammatory mediators and neuropeptides by immune and structural cells and nerve endings at the site of inflammation. Evidence showed that local and systemic NGF levels increase in patients with allergic conjunctivitis, including allergic rhinoconjuncivitis, vernal keratoconjunctivitis and atopic keratoconjunctivitis. We recently described an increase of conjunctival p75NTR expression in patients with allergic rhinoconjuncivitis, and an increase of tear levels of NGF after conjunctival provocation test with allergen. SUMMARY: NGF modulates ocular allergic reaction. Increasing understanding of the role of neuropeptides in allergic conjunctivitis may pave the way to the development of novel therapeutic approaches and improvement of patients' management.

Extracellular NGFR Spacers Allow Efficient Tracking and Enrichment of Fully Functional CAR-T Cells Co-Expressing a Suicide Gene.

Chimeric antigen receptor (CAR)-T cell immunotherapy is at the forefront of innovative cancer therapeutics. However, lack of standardization of cellular products within the same clinical trial and lack of harmonization between different trials have hindered the clear identification of efficacy and safety determinants that should be unveiled in order to advance the field. With the aim of facilitating the isolation and in vivo tracking of CAR-T cells, we here propose the inclusion within the CAR molecule of a novel extracellular spacer based on the low-affinity nerve-growth-factor receptor (NGFR). We screened four different spacer designs using as target antigen the CD44 isoform variant 6 (CD44v6). We successfully generated NGFR-spaced CD44v6 CAR-T cells that could be efficiently enriched with clinical-grade immuno-magnetic beads without negative consequences on subsequent expansion, immuno-phenotype, in vitro antitumor reactivity, and conditional ablation when co-expressing a suicide gene. Most importantly, these cells could be tracked with anti-NGFR monoclonal antibodies in NSG mice, where they expanded, persisted, and exerted potent antitumor effects against both high leukemia and myeloma burdens. Similar results were obtained with NGFR-enriched CAR-T cells specific for CD19 or CEA, suggesting the universality of this strategy. In conclusion, we have demonstrated that the incorporation of the NGFR marker gene within the CAR sequence allows for a single molecule to simultaneously work as a therapeutic and selection/tracking gene. Looking ahead, NGFR spacer enrichment might allow good manufacturing procedures-manufacturing of standardized CAR-T cell products with high therapeutic potential, which could be harmonized in different clinical trials and used in combination with a suicide gene for future application in the allogeneic setting.

Flow Cytometric Detection of Circulating Tumor Cells Using a Candidate Stem Cell Marker, p75 Neurotrophin Receptor (p75NTR).

The most widely studied detection for circulating tumor cells (CTCs) in peripheral blood of cancer patients has been based on immunomagnetic enrichment using antibodies against epithelial cell adhesion molecule (EpCAM), which is overexpressed in epithelial cells. A neurotrophin receptor p75 (p75NTR) is expressed in a candidate stem cell fraction in esophageal squamous cell carcinoma (ESCC), which shows significantly higher colony formation, enhanced tumor formation in mice, along with strong expression of epithelial mesenchymal transition-related genes. Here, we describe a method to detect CTCs in ESCC based on the combined expression of EpCAM and p75NTR using flow cytometry, demonstrating the feasibility of expression analysis of multiple cell surface markers in viable cells.

Inter-patient and Intra-tumor Heterogeneity in the Sensitivity to Tumor-targeted Immunity in Colorectal Cancer.

  Efficacy of immune checkpoint inhibitors such as PD-1 antibody for colorectal cancer remains to be proved except in microsatellite-instability-high (MSI-H) cases. While the objective response rate of MSI-H cases was 40%, that of microsatellite-stable (MSS) cases was 0%, showing that response rate to immune checkpoint inhibitors varies even among the microsatellite status. Some possible mechanisms that confer each patient variation in the response to immunotherapy should be considered. We focused on the combination of inter-patient heterogeneity and intra-tumor heterogeneity as a determining factor of immune reaction. An example of intra-tumor heterogeneity is the low expression of tumor antigen by CD271+ cells in melanoma. It is not surprising that similar mechanism exists in CRC. Other related intra-tumor heterogeneity includes EMT and autophagy, both molecular mechanisms that are thought to promote immune-evading phenotype. Besides the microsatellite status, inter-patient heterogeneity in response to tumor immunity includes hypermutator phenotype, which is driven by POLE mutation, intrinsic cytokine production, and microbiota in the gut.

Nerve Growth Factor Signaling from Membrane Microdomains to the Nucleus: Differential Regulation by Caveolins.

Membrane microdomains or "lipid rafts" have emerged as essential functional modules of the cell, critical for the regulation of growth factor receptor-mediated responses. Herein we describe the dichotomy between caveolin-1 and caveolin-2, structural and regulatory components of microdomains, in modulating proliferation and differentiation. Caveolin-2 potentiates while caveolin-1 inhibits nerve growth factor (NGF) signaling and subsequent cell differentiation. Caveolin-2 does not appear to impair NGF receptor trafficking but elicits prolonged and stronger activation of MAPK (mitogen-activated protein kinase), Rsk2 (ribosomal protein S6 kinase 2), and CREB (cAMP response element binding protein). In contrast, caveolin-1 does not alter initiation of the NGF signaling pathway activation; rather, it acts, at least in part, by sequestering the cognate receptors, TrkA and p75NTR, at the plasma membrane, together with the phosphorylated form of the downstream effector Rsk2, which ultimately prevents CREB phosphorylation. The non-phosphorylatable caveolin-1 serine 80 mutant (S80V), no longer inhibits TrkA trafficking or subsequent CREB phosphorylation. MC192, a monoclonal antibody towards p75NTR that does not block NGF binding, prevents exit of both NGF receptors (TrkA and p75NTR) from lipid rafts. The results presented herein underline the role of caveolin and receptor signaling complex interplay in the context of neuronal development and tumorigenesis.

Dermal CD271+ Cells are Closely Associated with Regeneration of the Dermis in the Wound Healing Process.

Stem cells have recently been shown to play important roles in wound healing. The aim of this study was to investigate the role of dermal CD271+ cells in wound healing. Full-thickness wounds were produced on the backs of 5-year-old and 24-week-old mice, and time-course of wound closure, CD271+ cell counts, and gene expression levels were compared. Delayed wound healing was observed in 24-week-old mice. The peak of CD271+ cell increase was delayed in 24-week-old mice, and gene expression levels of growth factors in wounded tissue were significantly increased in 5-year-old mice. Dermal CD271+ cells purified by fluorescence-activated cell sorting (FACS) expressed higher growth factors than CD271- cells, suggesting that CD271+ cells play important roles by producing growth factors. This study also investigated dermal CD271+ cells in patients with chronic skin ulcers. Dermal CD271+ cells in patients were significantly reduced compared with in healthy controls. Thus, dermal CD271+ cells are closely associated with wound healing.

p75NTR and Its Ligand ProNGF Activate Paracrine Mechanisms Etiological to the Vascular, Inflammatory, and Neurodegenerative Pathologies of Diabetic Retinopathy.

UNLABELLED: In many diseases, expression and ligand-dependent activity of the p75(NTR) receptor can promote pericyte and vascular dysfunction, inflammation, glial activation, and neurodegeneration. Diabetic retinopathy (DR) is characterized by all of these pathological events. However, the mechanisms by which p75(NTR) may be implicated at each stage of DR pathology remain poorly understood. Using a streptozotocin mouse model of diabetic retinopathy, we report that p75(NTR) is upregulated very early in glia and in pericytes to mediate ligand-dependent induction of inflammatory cytokines, disruption of the neuro-glia-vascular unit, promotion of blood-retina barrier breakdown, edema, and neuronal death. In a mouse model of oxygen-induced retinopathy, mimicking proliferative DR, p75(NTR)-dependent inflammation leads to ischemia and pathological angiogenesis through Semaphorin 3A. The acute use of antagonists of p75(NTR) or antagonists of the ligand proNGF suppresses each distinct phase of pathology, ameliorate disease, and prevent disease progression. Thus, our study documents novel disease mechanisms and validates druggable targets for diabetic retinopathy. SIGNIFICANCE STATEMENT: Diabetic retinopathy (DR) affects an estimated 250 million people and has no effective treatment. Stages of progression comprise pericyte/vascular dysfunction, inflammation, glial activation, and neurodegeneration. The pathophysiology of each stage remains unclear. We postulated that the activity of p75NTR may be implicated. We show that p75NTR in glia and in pericytes mediate ligand-dependent induction of inflammatory cytokines, disruption of the neuro-glia-vascular unit, promotion of blood-retina barrier breakdown, edema, and neuronal death. p75NTR-promoted inflammation leads to ischemia and angiogenesis through Semaphorin 3A. Antagonists of p75NTR or antagonists of proNGF suppress each distinct phase of pathology, ameliorate disease, and prevent disease progression. Our study documents novel mechanisms in a pervasive disease and validates druggable targets for treatment.

Correlation between nerve growth factor and tissue expression of IL-17 in leprosy.

Leprosy is a serious public health problem in peripheral and developing countries. Leprosy is a chronic infectious-contagious disease caused by the intracellular, bacillus Mycobacterium leprae, which causes tissue damage and demyelination of peripheral nerves. Recent studies have demonstrated the participation of new subtype's cytokines profile in the inflammatory response of leprosy. Since nerve functions are affected by inflammatory response during the course of leprosy, changes in the production of NGF and its receptor (NGF R) may be directly associated with disability and sensory loss. Skin biopsies were collected and submitted to immunohistochemistry using specific antibodies to IL-17, NGF and NGF R. Quantitative analysis of NGF, NGFR and IL-17 immunostaining showed a significant difference between the clinical forms, with higher expression of NGF and NGFR in lepromatous leprosy and IL-17 in tuberculoid leprosy. The present study showed that IL-17, in addition to stimulating an inflammatory response, negatively regulates the action of NGF and NGF R in the polar forms of the disease.

proBDNF and p75NTR Control Excitability and Persistent Firing of Cortical Pyramidal Neurons.

Persistent firing of entorhinal cortex (EC) pyramidal neurons is a key component of working and spatial memory. We report here that a pro-brain-derived neurotrophic factor (proBDNF)-dependent p75NTR signaling pathway plays a major role in excitability and persistent activity of pyramidal neurons in layer V of the EC. Using electrophysiological recordings, we show that proBDNF suppresses persistent firing in entorhinal slices from wild-type mice but not from p75NTR-null mice. Conversely, function-blocking proBDNF antibodies enhance excitability of pyramidal neurons and facilitate their persistent firing, and acute exposure to function-blocking p75NTR antibodies results in enhanced firing activity of pyramidal neurons. Genetic deletion of p75NTR specifically in neurons or during adulthood also induces enhanced excitability and persistent activity, indicating that the proBDNF-p75NTR signaling cascade functions within adult neurons to inhibit pyramidal activity. Phosphatidylinositol 4,5-bisphosphate (PIP2)-sensitive transient receptor potential canonical channels play a critical role in mediating persistent firing in the EC and we hypothesized that proBDNF-dependent p75NTR activation regulates PIP2 levels. Accordingly, proBDNF decreases cholinergic calcium responses in cortical neurons and affects carbachol-induced depletion of PIP2. Further, we show that the modulation of persistent firing by proBDNF relies on a p75NTR-Rac1-PI4K pathway. The hypothesis that proBDNF and p75NTR maintain network homeostasis in the adult CNS was tested in vivo and we report that p75NTR-null mice show improvements in working memory but also display an increased propensity for severe seizures. We propose that the proBDNF-p75NTR axis controls pyramidal neuron excitability and persistent activity to balance EC performance with the risk of runaway activity. SIGNIFICANCE STATEMENT: Persistent firing of entorhinal cortex (EC) pyramidal neurons is required for working memory. We report here that pro-brain-derived neurotrophic factor (proBDNF) activates p75NTR to induce a Rac1-dependent and phosphatidylinositol 4,5-bisphosphate-dependent signaling cascade that suppresses persistent activity. Conversely, using loss-of-function approaches, we find that endogenous proBDNF or p75NTR activation strongly decreases pyramidal neuron excitability and persistent firing, suggesting that a physiological role of this proBDNF-p75NTR cascade may be to regulate working memory in vivo. Consistent with this, mice rendered null for p75NTR during adulthood show improvements in working memory but also display an increased propensity for severe seizures. We propose that by attenuating EC network performance, the proBDNF-p75NTR signaling cascade reduces the probability of epileptogenesis.

Selection of a breast cancer subpopulation-specific antibody using phage display on tissue sections.

Breast cancer tumors are composed of heterogeneous cell populations. These populations display a high variance in morphology, growth and metastatic propensity. They respond differently to therapeutic interventions, and some may be more prone to cause recurrence. Studying individual subpopulations of breast cancer may provide crucial knowledge for the development of individualized therapy. However, this process is challenged by the availability of biomarkers able to identify subpopulations specifically. Here, we demonstrate an approach for phage display selection of recombinant antibody fragments on cryostat sections of human breast cancer tissue. This method allows for selection of recombinant antibodies binding to antigens specifically expressed in a small part of the tissue section. In this case, a CD271(+) subpopulation of breast cancer cells was targeted, and these may be potential breast cancer stem cells. We isolated an antibody fragment LH 7, which in immunohistochemistry experiments demonstrates specific binding to breast cancer subpopulations. The selection of antibody fragments directly on small defined areas within a larger section of malignant tissue is a novel approach by which it is possible to better target cellular heterogeneity in proteomic studies. The identification of novel biomarkers is relevant for our understanding and intervention in human diseases. The selection of the breast cancer-specific antibody fragment LH 7 may reveal novel subpopulation-specific biomarkers, which has the potential to provide new insight and treatment strategies for breast cancer.

An NCR1-based chimeric receptor endows T-cells with multiple anti-tumor specificities.

The Ral (Ras-like) GTP-binding proteins (RalA and RalB), as effectors of the proto-oncogene Natural killer (NK) cells are an important component of the anti-tumor response. Tumor recognition by NK cells was found to be partly triggered by molecules termed natural cytotoxic receptors (NCRs). Adoptive transfer of genetically-engineered tumor-reactive T-lymphocytes can mediate remarkable tumor regressions mostly in melanoma and leukemia patients. Yet, the application of such treatments to other cancers is needed and dependent on the isolation of receptors that could facilitate efficient recognition of these malignancies. Herein, we aimed at combining NK tumor recognition capability with the genetic modification of T-cells to provide the latter with a means to recognize several tumors in a non-MHC restricted way. Consequently, we generated and evaluated several chimeric receptors based on the extracellular domain of NCR1 (NKp46) fused to multiple signaling moieties and assess their antitumor activity when retrovirally expressed in T-cells. Following co-culture with different tumors, primary human T-lymphocytes expressing a chimeric NCR1 molecule recognized target cells derived from lung, cervical carcinoma, leukemia and pancreatic cancer. In addition, this receptor mediated an upregulation of surface activation markers and significant antitumor cytotoxicity both in vitro and in vivo. These results have meaningful implications for the immunotherapeutic treatment of cancer using gene-modified T-cells.