Neuropilins (NRPs) are non-tyrosine kinase cell surface glycoproteins expressed in all vertebrates and widely conserved across species. The two isoforms, such as neuropilin-1 (NRP1) and neuropilin-2 (NRP2), mainly act as coreceptors for class III Semaphorins and for members of the vascular endothelial growth factor family of molecules and are widely known for their role in a wide array of physiological processes, such as cardiovascular, neuronal development and patterning, angiogenesis, lymphangiogenesis, as well as various clinical disorders. Intriguingly, additional roles for NRPs occur with myeloid and lymphoid cells, in normal physiological as well as different pathological conditions, including cancer, immunological disorders, and bone diseases. However, little is known concerning the molecular pathways that govern these functions. In addition, NRP1 expression has been characterized in different immune cellular phenotypes including macrophages, dendritic cells, and T cell subsets, especially regulatory T cell populations. By contrast, the functions of NRP2 in immune cells are less well known. In this review, we briefly summarize the genomic organization, structure, and binding partners of the NRPs and extensively discuss the recent advances in their role and function in different immune cell subsets and their clinical implications.

Tumor-associated macrophages (TAM) are causally associated with tumorigenesis as well as regulation of antitumor immune responses and have emerged as potential immunotherapeutic targets. Recent evidence suggests TAM phagocytose apoptotic tumor cells within the tumor microenvironment through efferocytosis in an immunologically silent manner, thus maintaining an immunosuppressed microenvironment. The signal transduction pathways coupling efferocytosis and immunosuppression are not well known. Neuropilin-2 (NRP2) is a member of the membrane-associated neuropilin family and has been reported in different immune cells but is poorly characterized. In this study, we show that NRP2 is expressed during macrophage differentiation, is induced by tumor cells, and regulates phagocytosis in macrophages. Furthermore, NRP2 in TAM promoted efferocytosis and facilitated tumor growth. Deletion of NRP2 from TAM impaired the clearance of apoptotic tumor cells and increased secondary necrosis within tumors. This resulted in a break in the immune tolerance and reinitiated antitumor immune responses, characterized by robust infiltration of CD8 T and natural killer cells. This result suggests NRP2 may act as a molecular mediator that connects efferocytosis and immune suppression. Deletion of NRP2 in TAM downregulated several immunosuppressive and tumor-promoting genes and upregulated immunostimulatory genes in the myeloid compartment. Taken together, our study demonstrates that TAM-derived NRP2 plays a crucial role in tumor promotion through efferocytosis, opening the enticing option for the development of effective immunotherapy targeting TAM. Neuropilin-2 in macrophages promotes tumor growth by regulating efferocytosis of apoptotic tumor cells and orchestrating immune suppression. http://cancerres.aacrjournals.org/content/canres/78/19/5600/F1.large.jpg .

BackgroundVisceral leishmaniasis (VL) is a deadly parasitic diseases caused by Leishmania donovani; it is a major health problem in many countries. A lack of proper understanding of the disease biology, poor diagnostic methods and increasing drug resistance are the main reasons for the growing burden of VL infection. Comparative plasma proteomics are a relatively useful technique that can be used to investigate disease-associated alterations that can help in understanding host responses against pathogens, and might be useful in disease management and diagnosis.ResultIn this study, a comparative proteomics and glycoproteomics approach using 2DE and 2D-DIGE was employed between early diagnosed VL patients of all age groups and healthy endemic and non-endemic controls in order to aid the recognition of disease-associated alterations in host plasma. Comparative proteomics was performed by the depletion of seven highly abundant plasma proteins. Comparative glycoproteomics was performed by the depletion of albumin and IgG, followed by purification of plasma glycoproteins using a multi lectin affinity column. From these two approaches, 39 differentially expressed protein spots were identified and sequenced using MALDI-TOF/TOF mass spectrometry. This revealed ten distinct proteins that appeared in multiple spots, suggesting micro-heterogeneity. Among these proteins, alpha-1-antitrypsin, alpha-1-B glycoprotein and amyloid-A1 precursor were up-regulated, whereas vitamin-D binding protein, apolipoprotein-A-I and transthyretin were down-regulated in VL. Alterations in the levels of these proteins in VL-infected plasma were further confirmed by western blot and ELISA.ConclusionsThese proteins may be involved in the survival of parasites, resisting neutrophil elastase, and in their multiplication in macrophages, potentially maintaining endogenous anti-inflammatory and immunosuppressive conditions. Consequently, the results of this study may help in understanding the host response against L.donovani, which could help in the discovery of new drugs and disease management. Finally, these alterations on protein levels might be beneficial in improving early diagnosis considering those as biomarkers in Indian VL.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-014-0048-z) contains supplementary material, which is available to authorized users.

As malignant transformation requires synchronization of growth-driving signaling (S) and metabolic (M) pathways, defining cancer-specific S-M interconnected networks (SMINs) could lead to better understanding of oncogenic processes. In a systems-biology approach, we developed a mathematical model for SMINs in mutated EGF receptor (EGFRvIII) compared to wild-type EGF receptor (EGFRwt) expressing glioblastoma multiforme (GBM). Starting with experimentally validated human protein-protein interactome data for S-M pathways, and incorporating proteomic data for EGFRvIII and EGFRwt GBM cells and patient transcriptomic data, we designed a dynamic model for EGFR-driven GBM-specific information flow. Key nodes and paths identified by in silico perturbation were validated experimentally when inhibition of signaling pathway proteins altered expression of metabolic proteins as predicted by the model. This demonstrated capacity of the model to identify unknown connections between signaling and metabolic pathways, explain the robustness of oncogenic SMINs, predict drug escape, and assist identification of drug targets and the development of combination therapies.

BackgroundAccumulation of regulatory T cells (Treg) has been described to often correlate with poor prognosis in many solid tumors. How Treg presence impinges on limited functionality and clonal composition of tumor-associated CD8 +T cells has important implications for their therapeutic targeting in the tumor microenvironment. In the present study, we investigated how accumulation of Tregs contributes to T cell dysfunction and clonal constriction of tumor-infiltrating CD8 +T cells.MethodsResected melanoma and lung adenocarcinoma tissues from tumor-bearing mice or patients were analyzed. The proportions and phenotype as well as clonal diversity of tumor-associated CD8 +T cells were evaluated by flow cytometry and single-cell T-cell receptor (TCR) sequencing, respectively, at early or advanced tumor stages or under Treg depletion conditions. Furthermore, antigen-specific T cells were evaluated on adoptive transfer into tumor-bearing mice in the presence or absence of anti-CTLA-4 antibody or CTLA-4 Ig. Lastly, tumor-bearing mice were treated with anti-KLRG1 antibody and/or bromodomain inhibitor JQ1 with interleukin (IL)-2 immune complexes to determine therapeutic efficacy.ResultsWe demonstrate that the emergence of exhaustion-like phenotype and impaired effector functionality in tumor-associated CD8 +T cells is positively correlated with Treg accumulation in the tumor bed and this dysfunctional phenotype becomes reversed on Treg reduction in murine melanoma and lung cancer models. Heightened tumor-associated Treg-expressed CTLA-4 is key to emergence and sustenance of this phenotype. Furthermore, TCR sequencing revealed a clonal shrinkage of tumor-infiltrating CD8 +T cells as tumor progressed, which was associated with reduced survival profile concomitant to increasing Treg proportions. Limited IL-2 availability was a key mechanism contributing to this peripheral repertoire reshaping as Treg depletion improved IL-2 levels, rescued CD8 +T cell viability, and improved their clonal diversity. Finally, targeted reduction of tumor but not peripheral Tregs through JQ1 and/or anti-KLRG1 antibody significantly improved antitumor response in melanoma-bearing mice when supplemented with IL-2 immune complexes.ConclusionCollectively, our study reveals a bimodal program enacted by Tregs to support T cell dysfunction in the tumor bed and highlights a promising therapeutic regimen for localized reprogramming of the tumor microenvironment to curb Treg impairment of antitumor CD8 +T cell response in favor of improved antitumor immunity.

BackgroundFor a long time cancer cells are known for increased uptake of glucose and its metabolization through glycolysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key regulatory enzyme of this pathway and can produce ATP through oxidative level of phosphorylation. Previously, we reported that GAPDH purified from a variety of malignant tissues, but not from normal tissues, was strongly inactivated by a normal metabolite, methylglyoxal (MG). Molecular mechanism behind MG mediated GAPDH inhibition in cancer cells is not well understood.MethodsGAPDH was purified from Ehrlich ascites carcinoma (EAC) cells based on its enzymatic activity. GAPDH associated proteins in EAC cells and 3-methylcholanthrene (3MC) induced mouse tumor tissue were detected by mass spectrometry analysis and immunoprecipitation (IP) experiment, respectively. Interacting domains of GAPDH and its associated proteins were assessed by in silico molecular docking analysis. Mechanism of MG mediated GAPDH inactivation in cancer cells was evaluated by measuring enzyme activity, Circular dichroism (CD) spectroscopy, IP and mass spectrometry analyses.ResultHere, we report that GAPDH is associated with glucose-6-phosphate isomerase (GPI) and pyruvate kinase M2 (PKM2) in Ehrlich ascites carcinoma (EAC) cells and also in 3-methylcholanthrene (3MC) induced mouse tumor tissue. Molecular docking analyses suggest C-terminal domain preference for the interaction between GAPDH and GPI. However, both C and N termini of PKM2 might be interacting with the C terminal domain of GAPDH. Expression of both PKM2 and GPI is increased in 3MC induced tumor compared with the normal tissue. In presence of 1 mM MG, association of GAPDH with PKM2 or GPI is not perturbed, but the enzymatic activity of GAPDH is reduced to 26.8 ± 5 % in 3MC induced tumor and 57.8 ± 2.3 % in EAC cells. Treatment of MG to purified GAPDH complex leads to glycation at R399 residue of PKM2 only, and changes the secondary structure of the protein complex.ConclusionPKM2 may regulate the enzymatic activity of GAPDH. Increased enzymatic activity of GAPDH in tumor cells may be attributed to its association with PKM2 and GPI. Association of GAPDH with PKM2 and GPI could be a signature for cancer cells. Glycation at R399 of PKM2 and changes in the secondary structure of GAPDH complex could be one of the mechanisms by which GAPDH activity is inhibited in tumor cells by MG.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2172-x) contains supplementary material, which is available to authorized users.

Understanding the role of neuropilin 2 (NRP2) in prostate cancer cells as well as in the bone microenvironment is pivotal in the development of an effective targeted therapy for the treatment of prostate cancer bone metastasis. We observed a significant upregulation of NRP2 in prostate cancer cells metastasized to bone. Here, we report that targeting NRP2 in cancer cells can enhance taxane-based chemotherapy with a better therapeutic outcome in bone metastasis, implicating NRP2 as a promising therapeutic target. Since, osteoclasts present in the tumor microenvironment express NRP2, we have investigated the potential effect of targeting NRP2 in osteoclasts. Our results revealed NRP2 negatively regulates osteoclast differentiation and function in the presence of prostate cancer cells that promotes mixed bone lesions. Our study further delineated the molecular mechanisms by which NRP2 regulates osteoclast function. Interestingly, depletion of NRP2 in osteoclasts in vivo showed a decrease in the overall prostate tumor burden in the bone. These results therefore indicate that targeting NRP2 in prostate cancer cells as well as in the osteoclastic compartment can be beneficial in the treatment of prostate cancer bone metastasis.

The literature surrounding KLRG1 has primarily focused on NK and CD8+ T cells. However, there is evidence that the most suppressive Tregs express KLRG1. Until now, the role of KLRG1 on Tregs has been mostly overlooked and remains to be elucidated. Here we review the current literature on KLRG1 with an emphasis on the KLRG1+ Treg subset role during cancer development and autoimmunity. KLRG1 has been recently proposed as a new checkpoint inhibitor target, but these studies focused on the effects of KLRG1 blockade on effector cells. We propose that when designing anti-tumor therapies targeting KLRG1, the effects on both effector cells and Tregs will have to be considered.