Neem Leaf Glycoprotein disrupts exhausted CD8+ T cell-mediated cancer stem cell aggression.

Targeting exhausted CD8+T cell (TEX) induced aggravated cancer stem cells (CSC) holds immense therapeutic potential. In this regard, immunomodulation via Neem Leaf Glycoprotein (NLGP), a plant-derived glycoprotein immunomodulator is explored. Since former reports have proven immune-dependent tumor restriction of NLGP across multiple tumor models, we hypothesized that NLGP might reprogram and rectify TEX to target CSCs successfully. Here we report that NLGP's therapeutic administration significantly reduced TEX -associated CSC virulence in in vivo B16-F10 melanoma tumor model. Similar trend was observed in in vitro generated TEX and B16-F10/MCF7 co-culture setups. NLGP rewired CSCs by downregulating clonogenicity, multidrug resistance phenotypes and PDL1, OCT4, SOX2 expression. Cell cycle analysis revealed that NLGP-educated TEX efficiently pushed CSCs out of quiescent-phase (G0G1) into synthesis-phase (S), supported by hyper-phosphorylation of G0G1-S transitory cyclins and Rb-proteins. This rendered quiescent CSCs susceptible to s-phase targeting chemotherapeutic drugs like 5-Fluorouracil (5FU). Consequently combinatorial treatment of NLGP and 5FU brought optimal CSC targeting efficiency with increase in apoptotic bodies and pro-apoptotic BID expression. Notably a strong nephron-protective effect of NLGP was also observed, which prevented 5FU associated toxicity. Furthermore, Dectin-1 mediated NLGP uptake and subsequent alteration of Notch1 and mTOR axis was deciphered as the involved signalling network. This observation unveiled Dectin-1 as a potent immunotherapeutic drug-target to counter T cell exhaustion. Cumulatively, NLGP immunotherapy alleviated exhausted CD8+T cell induced CSC aggravation. Implications: Our study recommends that NLGP-immunotherapy can be utilized to counter ramifications of T cell exhaustion and to target therapy elusive aggressive CSCs without evoking toxicity.

The immunomodulatory impact of naturally derived neem leaf glycoprotein on the initiation progression model of 4NQO induced murine oral carcinogenesis: a preclinical study.

Introduction: Murine tumor growth restriction by neem leaf glycoprotein (NLGP) was established in various transplanted models of murine sarcoma, melanoma and carcinoma. However, the role of NLGP in the sequential carcinogenic steps has not been explored. Thus, tongue carcinogenesis in Swiss mice was induced by 4-nitroquinoline-1-oxide (4NQO), which has close resemblance to human carcinogenesis process. Interventional role of NLGP in initiation-promotion protocol established during 4NQO mediated tongue carcinogenesis in relation to systemic immune alteration and epithelial-mesenchymal transition (EMT) is investigated. Methods: 4NQO was painted on tongue of Swiss mice every third day at a dose of 25µl of 5mg/ml stock solution. After five consecutive treatment with 4NQO (starting Day7), one group of mice was treated with NLGP (s.c., 25µg/mice/week), keeping a group as PBS control. Mice were sacrificed in different time-intervals to harvest tongues and studied using histology, immunohistochemistry, flow-cytometry and RT-PCR on different immune cells and EMT markers (e-cadherin, vimentin) to elucidate their phenotypic and secretory status. Results: Local administration of 4NQO for consecutive 300 days promotes significant alteration in tongue mucosa including erosion in papillae and migration of malignant epithelial cells to the underlying connective tissue stroma with the formation of cell nests (exophytic-hyperkeratosis with mild dysplasia). Therapeutic NLGP treatment delayed pre-neoplastic changes promoting normalization of mucosa by maintaining normal structure. Flow-cytometric evidences suggest that NLGP treatment upregulated CD8+, IFNγ+, granzyme B+, CD11c+ cells in comparison to 4NQO treated mice with a decrease in Ki67+ and CD4+FoxP3+ cells in NLGP treated cohort. RT-PCR demonstrated a marked reduction of MMP9, IL-6, IL-2, CD31 and an upregulation in CCR5 in tongues from 4NQO+NLGP treated mice in comparison to 4NQO treated group. Moreover, 4NQO mediated changes were associated with reduction of e-cadherin and simultaneous up-regulation of vimentin expression in epithelium that was partially reversed by NLGP. Discussion: Efficacy of NLGP was tested first time in sequential carcinogenesis model and proved effective in delaying the initial progression. NLGP normalizes type 1 immunity including activation of the CD8+T effector functions, reduction of regulatory T cell functions, along with changes in EMT to make the host systemically alert to combat the carcinogenic threat.

Neem leaf glycoprotein binding to Dectin-1 receptors on dendritic cell induces type-1 immunity through CARD9 mediated intracellular signal to NFκB.

BACKGROUND: A water-soluble ingredient of mature leaves of the tropical mahogany 'Neem' (Azadirachta indica), was identified as glycoprotein, thus being named as 'Neem Leaf Glycoprotein' (NLGP). This non-toxic leaf-component regressed cancerous murine tumors (melanoma, carcinoma, sarcoma) recurrently in different experimental circumstances by boosting prime antitumor immune attributes. Such antitumor immunomodulation, aid cytotoxic T cell (Tc)-based annihilation of tumor cells. This study focused on identifying and characterizing the signaling gateway that initiate this systemic immunomodulation. In search of this gateway, antigen-presenting cells (APCs) were explored, which activate and induce the cytotoxic thrust in Tc cells. METHODS: Six glycoprotein-binding C-type lectins found on APCs, namely, MBR, Dectin-1, Dectin-2, DC-SIGN, DEC205 and DNGR-1 were screened on bone marrow-derived dendritic cells from C57BL/6 J mice. Fluorescence microscopy, RT-PCR, flow cytometry and ELISA revealed Dectin-1 as the NLGP-binding receptor, followed by verifications through RNAi. Following detection of ß-Glucans in NLGP, their interactions with Dectin-1 were explored in silico. Roles of second messengers and transcription factors in the downstream signal were studied by co-immunoprecipitation, western blotting, and chromatin-immunoprecipitation. Intracellularization of FITC-coupled NLGP was observed by processing confocal micrographs of DCs. RESULTS: Considering extents of hindrance in NLGP-driven transcription rates of the cytokines IL-10 and IL-12p35 by receptor-neutralization, Dectin-1 receptors on dendritic cells were found to bind NLGP through the ligand's peripheral ß-Glucan chains. The resulting signal phosphorylates PKCδ, forming a trimolecular complex of CARD9, Bcl10 and MALT1, which in turn activates the canonical NFκB-pathway of transcription-regulation. Consequently, the NFκB-heterodimer p65:p50 enhances Il12a transcription and the p50:p50 homodimer represses Il10 transcription, bringing about a cytokine-based systemic-bias towards type-1 immune environment. Further, NLGP gets engulfed within dendritic cells, possibly through endocytic activities of Dectin-1. CONCLUSION: NLGP's binding to Dectin-1 receptors on murine dendritic cells, followed by the intracellular signal, lead to NFκB-mediated contrasting regulation of cytokine-transcriptions, initiating a pro-inflammatory immunopolarization, which amplifies further by the responding immune cells including Tc cells, alongside their enhanced cytotoxicity. These insights into the initiation of mammalian systemic immunomodulation by NLGP at cellular and molecular levels, may help uncovering its mode of action as a novel immunomodulator against human cancers, following clinical trials.

Cancer stem cell-immune cell crosstalk in breast tumor microenvironment: a determinant of therapeutic facet.

Breast cancer (BC) is globally one of the leading killers among women. Within a breast tumor, a minor population of transformed cells accountable for drug resistance, survival, and metastasis is known as breast cancer stem cells (BCSCs). Several experimental lines of evidence have indicated that BCSCs influence the functionality of immune cells. They evade immune surveillance by altering the characteristics of immune cells and modulate the tumor landscape to an immune-suppressive type. They are proficient in switching from a quiescent phase (slowly cycling) to an actively proliferating phenotype with a high degree of plasticity. This review confers the relevance and impact of crosstalk between immune cells and BCSCs as a fate determinant for BC prognosis. It also focuses on current strategies for targeting these aberrant BCSCs that could open avenues for the treatment of breast carcinoma.

Toll-like receptor 2 selectively modulates Ras isoforms expression in Leishmania major infection.

Leishmania infection of macrophages results in altered Ras isoforms expression and Toll-like receptor-2 (TLR2) expression and functions. Therefore, we examined whether TLR2 would selectively alter Ras isoforms' expression in macrophages. We observed that TLR2 ligands- Pam3CSK4, peptidoglycan (PGN), and FSL- selectively modulated the expression of Ras isoforms in BALB/c-derived elicited macrophages. Lentivirally-expressed TLR1-shRNA significantly reversed this Ras isoforms expression profile. TLR2-deficient L. major-infected macrophages and the lymph node cells from the L. major-infected mice showed similarly reversed Ras isoforms expression. Transfection of the macrophages with the siRNAs for the adaptors- Myeloid Differentiation factor 88 (MyD88) and Toll-Interleukin-1 Receptor (TIR) domain-containing adaptor protein (TIRAP)- or Interleukin-1 Receptor-Associated Kinases (IRAKs)- IRAK1 and IRAK4- significantly inhibited the L. major-induced down-regulation of K-Ras, and up-regulation of N-Ras and H-Ras, expression. The TLR1/TLR2-ligand Pam3CSK4 increased IL-10 and TGF-ß expression in macrophages. Pam3CSK4 upregulated N-Ras and H-Ras, but down-regulated K-Ras, expression in C57BL/6 wild-type, but not in IL-10-deficient, macrophages. IL-10 or TGF-ß signaling inhibition selectively regulated Ras isoforms expression. These observations indicate the specificity of the TLR2 regulation of Ras isoforms and their selective modulation by MyD88, TIRAP, and IRAKs, but not IL-10 or TGF-ß, signaling.

Terminally Exhausted CD8+ T Cells Resistant to PD-1 Blockade Promote Generation and Maintenance of Aggressive Cancer Stem Cells.

SIGNIFICANCE: Cross-talk with TTEX CD8+ T cells mediated by the VEGFR2 axis induces aggressive properties in cancer stem cells to promote tumor progression.

Evolvability and emergence of tumor heterogeneity as a space-time function.

The loss of control of cell proliferation, apoptosis regulation and contact inhibition leads to tumor development. While benign tumors are restricted to their primary space, i.e. where these tumors first originate, the metastatic tumors not only disseminate- facilitated by hypoxia-driven neovascularization- to distant secondary sites but also show substantial changes in metabolism, tissue architectures, gene expression profiles and immune phenotypes. All these alterations result in radio-, chemo- and immune-resistance rendering these metastatic tumor cells refractory to therapy. Since the beginning of the transformation, these factors- which influence each other- are incorporated to the developing and metastasizing tumor. As a result, the complexities in the heterogeneity of tumor progressively increase. This space-time function in the heterogeneity of tumors is generated by various conditions and factors at the genetic as well as microenvironmental levels, for example, endogenous retroviruses, methylation and epigenetic dysregulation that may be etiology-specific, cancer associated inflammation, remodeling of the extracellular matrix and mesenchymal cell shifted functions. On the one hand, these factors may cause de-differentiation of the tumor cells leading to cancer stem cells that contribute to radio-, chemo- and immune-resistance and recurrence of tumors. On the other hand, they may also enhance the heterogeneity under specific microenvironment-driven proliferation. In this editorial, we intend to underline the importance of heterogeneity in cancer progress, its evaluation and its use in correlation with the tumor evolution in a specific patient as a field of research for achieving precise patient-tailored treatments and amelioration of diagnostic (monitoring) tools and prognostic capacity.

High monocytic MDSC signature predicts multi-drug resistance and cancer relapse in non-Hodgkin lymphoma patients treated with R-CHOP.

Introduction: Non-Hodgkin Lymphoma (NHL) is a heterogeneous lymphoproliferative malignancy with B cell origin. Combinatorial treatment of rituximab, cyclophsphamide, hydroxydaunorubicin, oncovin, prednisone (R-CHOP) is the standard treatment regimen for NHL, yielding a complete remission (CR) rate of 40-50%. Unfortunately, considerable patients undergo relapse after CR or initial treatment, resulting in poor clinical implications. Patient's response to chemotherapy varies widely from static disease to cancer recurrence and later is primarily associated with the development of multi-drug resistance (MDR). The immunosuppressive cells within the tumor microenvironment (TME) have become a crucial target for improving the therapy efficacy. However, a better understanding of their involvement is needed for distinctive response of NHL patients after receiving chemotherapy to design more effective front-line treatment algorithms based on reliable predictive biomarkers. Methods: Peripheral blood from 61 CD20+ NHL patients before and after chemotherapy was utilized for immunophenotyping by flow-cytometry at different phases of treatment. In-vivo and in-vitro doxorubicin (Dox) resistance models were developed with murine Dalton's lymphoma and Jurkat/Raji cell-lines respectively and impact of responsible immune cells on generation of drug resistance was studied by RT-PCR, flow-cytometry and colorimetric assays. Gene silencing, ChIP and western blot were performed to explore the involved signaling pathways. Results: We observed a strong positive correlation between elevated level of CD33+CD11b+CD14+CD15- monocytic MDSCs (M-MDSC) and MDR in NHL relapse cohorts. We executed the role of M-MDSCs in fostering drug resistance phenomenon in doxorubicin-resistant cancer cells in both in-vitro, in-vivo models. Moreover, in-vitro supplementation of MDSCs in murine and human lymphoma culture augments early expression of MDR phenotypes than culture without MDSCs, correlated well with in-vitro drug efflux and tumor progression. We found that MDSC secreted cytokines IL-6, IL-10, IL-1ß are the dominant factors elevating MDR expression in cancer cells, neutralization of MDSC secreted IL-6, IL-10, IL-1ß reversed the MDR trait. Moreover, we identified MDSC secreted IL-6/IL-10/IL-1ß induced STAT1/STAT3/NF-κß signaling axis as a targeted cascade to promote early drug resistance in cancer cells. Conclusion: Our data suggests that screening patients for high titre of M-MDSCs might be considered as a new potential biomarker and treatment modality in overcoming chemo-resistance in NHL patients.

Tumor activated platelets induce vascular mimicry in mesenchymal stem cells and aid metastasis.

Extent of metastasis influences activation of platelets in tumor-microenvironment. Activated platelets potentiate mesenchymal-stem-cells (MSCs) to migrate in secondary metastatic sites without participation in process of invasion. Presence of higher percentage of MSCs along with activated-platelets induces formation of vascular-mimicry (VM). The pathophysiology, VM, has already been reported in multiple types of cancer including lung, ovary, melanoma etc. and related to poor-prognosis. Interaction of MSCs with platelets in cell-to-cell contact dependent manner is essential for their migration, thereby, VM. Evidences are obtained suggesting that under influence of tumor-associated-activated-platelets, expressions of vimentin, ve-cadherin are increased, along with decrease in e-cadherin on CD105+ MSCs in both mRNA and protein levels that may help in formation of vessel like structure in VM. Adoptive transfer of MSCs along with tumor-activated-platelets causes greater B16 melanoma metastasis at lungs in comparison to MSCs with non-activated platelets. Presence of CD105+Vimentin+ MSCs in vessel like structure in the metastatic lung confirms the involvement of platelet-activated-MSCs in VM, thereby, in metastasis.

COVID-19 imparted immune manifestation can be combated by NLGP: Lessons from cancer research.

SARS-CoV-2 easily infects human monocytes, macrophages and possibly dendritic cells (DCs), causing dysfunctions of these important antigen presenting cells (APCs). Observed DC dysfunctions facilitate improper antigen presentation, which obviously results T cell anergy, exhaustion and apoptosis, thus, may be contributing significantly in SARS-CoV-2 infection associated lymphopenia. Neem Leaf Glycoprotein or NLGP has enormous role in altered DC functions, thereby, offering optimum T cell mediated cytotoxicity, as experienced from cancer system. Such NLGP guided correction of altered DCs might also be effective to generate proper SARS-CoV-2-specific effector and central memory T cells.

NLGP regulates RGS5-TGFß axis to promote pericyte-dependent vascular normalization during restricted tumor growth.

Altered RGS5-associated intracellular pericyte signaling and its abnormal crosstalk with endothelial cells (ECs) result chaotic tumor-vasculature, prevent effective drug delivery, promote immune-evasion and many more to ensure ultimate tumor progression. Moreover, the frequency of lethal-RGS5high  pericytes within tumor was found to increase with disease progression, which signifies the presence of altered cell death pathway within tumor microenvironment (TME). In this study, we checked whether and how neem leaf glycoprotein (NLGP)-immunotherapy-mediated tumor growth restriction is associated with modification of pericytes' signaling, functions and its interaction with ECs. Analysis of pericytes isolated from tumors of NLGP treated mice suggested that NLGP treatment promotes apoptosis of NG2+ RGS5high -fuctionally altered pericytes by downregulating intra-tumoral TGFß, along with maintenance of more matured RGS5neg  pericytes. NLGP-mediated inhibition of TGFß within TME rescues binding of RGS5 with Gαi and thereby termination of PI3K-AKT mediated survival signaling by downregulating Bcl2 and initiating pJNK mediated apoptosis. Limited availability of TGFß also prevents complex-formation between RGS5 and Smad2 and rapid RGS5 nuclear translocation to mitigate alternate immunoregulatory functions of RGS5high  tumor-pericytes. We also observed binding of Ang1 from pericytes with Tie2 on ECs in NLGP-treated tumor, which support re-association of pericytes with endothelium and subsequent vessel stabilization. Furthermore, NLGP-therapy- associated RGS5 deficiency relieved CD4+  and CD8+ T cells from anergy by regulating 'alternate-APC-like' immunomodulatory characters of tumor-pericytes. Taken together, present study described the mechanisms of NLGP's effectiveness in normalizing tumor-vasculature by chiefly modulating pericyte-biology and EC-pericyte interactions in tumor-host to further strengthen its translational potential as single modality treatment.

TLR induced IL-27 plays host-protective role against B16BL6 melanoma in C57BL/6 mice.

Elicitation of the tumor-eliminating immune response is a major challenge, as macrophages- constituting a major component of solid tumor mass- play important roles in development, maintenance and tumor regression. The macrophage-expressed Toll-Like Receptors (TLRs) enhance macrophage function and their ability to activate T cells via secretion of cytokines, which may help in tumor regression. IL-27, a member of the IL-12 family of cytokines, is shown to exhibit anti-tumor and anti-angiogenic activities. Herein, we developed B16BL6 melanoma model in C75BL/6 mouse to dissect the crosstalk between TLRs and IL-27 in tumors. We report existence of a novel TLR- IL-27 feed-forward loop, whereby TLRs and IL-27 up-regulated each other's expression, which we found perturbed during melanoma tumorigenesis. Intra-tumoral injection of Imiquimod, a TLR7/8 ligand, reduced the tumor burden; the anti-tumor effect was reversed upon IL-27 and IL-27R silencing by intra-tumorally administered, lentivirally expressed IL-27 and IL-27R shRNA. The reduced tumor growth was accompanied by significantly fewer Treg cells but increased IFN-γ and granzyme B expression by CD8+ T cells. These data indicate the preventative role for TLR-induced IL-27 in aggressive and highly invasive melanoma.

Lactic acid in alternative polarization and function of macrophages in tumor microenvironment.

In developing tumor, macrophages are one major immune infiltrate that not only contributes in shaping up of tumor microenvironment (TME) but also have the potential of determining the fate of tumor in terms of its progression. Phenotypic plasticity of macrophages primarily channelizes them to alternative (M2) form of tumor associated macrophages (TAM) in the TME. One of the key tumor derived components that plays a crucial role in TAM polarization from M1 to M2 form is lactic acid and has prominent role in progression of malignancy. The role of lactic acid as signalling molecule as well as an immunomodulator has recently been recognized. This review focuses on the mechanism and signalling that are involved in lactic acid induced M2 polarization and possible therapeutic strategies for regulating lactic acidosis in TME.

Macrophages in tumor: An inflammatory perspective.

Inflammation is a part of carefully co-ordinated healing immune exercise to eliminate injurious stimuli. However, in substantial number of cancer types, it contributes in shaping up of robust tumor microenvironment (TME). Solid TME promotes infiltration of tumor associated macrophages (TAMs) that contributes to cancer promotion. TAMs are functionally heterogeneous and display an extraordinary degree of plasticity, which allow 'Switching' of macrophages into an 'M2', phenotype, linked with immunosuppression, advancement of tumor angiogenesis with metastatic consequences. In contrary to the classical M1 macrophages, these M2 TAMs are high-IL-10, TGF-ß secreting-'anti-inflammatory'. In this review, we will discuss the modes of infiltration and switching of TAMs into M2 anti-inflammatory state in the TME to promote immunosuppression and inflammation-driven cancer.

RGS5-TGFß-Smad2/3 axis switches pro- to anti-apoptotic signaling in tumor-residing pericytes, assisting tumor growth.

Regulator-of-G-protein-signaling-5 (RGS5), a pro-apoptotic/anti-proliferative protein, is a signature molecule of tumor-associated pericytes, highly expressed in several cancers, and is associated with tumor growth and poor prognosis. Surprisingly, despite the negative influence of intrinsic RGS5 expression on pericyte survival, RGS5highpericytes accumulate in progressively growing tumors. However, responsible factor(s) and altered-pathway(s) are yet to report. RGS5 binds with Gαi/q and promotes pericyte apoptosis in vitro, subsequently blocking GPCR-downstream PI3K-AKT signaling leading to Bcl2 downregulation and promotion of PUMA-p53-Bax-mediated mitochondrial damage. However, within tumor microenvironment (TME), TGFß appeared to limit the cytocidal action of RGS5 in tumor-residing RGS5highpericytes. We observed that in the presence of high RGS5 concentrations, TGFß-TGFßR interactions in the tumor-associated pericytes lead to the promotion of pSmad2-RGS5 binding and nuclear trafficking of RGS5, which coordinately suppressed RGS5-Gαi/q and pSmad2/3-Smad4 pairing. The RGS5-TGFß-pSmad2 axis thus mitigates both RGS5- and TGFß-dependent cellular apoptosis, resulting in sustained pericyte survival/expansion within the TME by rescuing PI3K-AKT signaling and preventing mitochondrial damage and caspase activation. This study reports a novel mechanism by which TGFß fortifies and promotes survival of tumor pericytes by switching pro- to anti-apoptotic RGS5 signaling in TME. Understanding this altered RGS5 signaling might prove beneficial in designing future cancer therapy.

Neem leaf glycoprotein salvages T cell functions from Myeloid-derived suppressor cells-suppression by altering IL-10/STAT3 axis in melanoma tumor microenvironment.

Myeloid-derived suppressor cells (MDSCs) suppress antitumor immune functions. We have observed that an immunomodulator, neem leaf glycoprotein (NLGP), inhibits tumor-resident MDSCs and enhances antitumor CD8+ T cell immunity. NLGP inhibits the number as well as functions of tumor-resident MDSCs (Gr1±CD11b±) and enhances antitumor CD8± T cell immunity by downregulating arginase 1 and inducible nitric oxide synthase production in MDSCs. Accordingly, decreased T cell anergy and helper to regulatory T cell conversion have been observed in the presence of NLGP, which ultimately augments T cell functions. Mechanistically, NLGP-mediated rectification of T cell suppressive functions of MDSCs was primarily associated with downregulation of the interleukin (IL)-10/signal transducer and activator of transcription 3 (STAT3) signaling axis within the tumor microenvironment, as confirmed by knockdown of STAT3 (by STAT3-siRNA) and using IL-10-/- mice. Thus, NLGP-mediated suppression of MDSC functions in tumor hosts is appeared to be another associated effective mechanism for the eradication of murine melanoma by NLGP.

Neem leaf glycoprotein reverses tumor-induced and age-associated thymic involution to maintain peripheral CD8+ T cell pool.

Aim: As tumor causes atrophy in the thymus to target effector-T cells, this study is aimed to decipher the efficacy of neem leaf glycoprotein (NLGP) in tumor- and age-associated thymic atrophy. Materials & methods: Different thymus parameters were studied using flow cytometry, reverse transcriptase PCR and immunocyto-/histochemistry in murine melanoma and sarcoma models. Results: Longitudinal NLGP therapy in tumor hosts show tumor-reduction along with significant normalization of thymic alterations. NLGP downregulates intrathymic IL-10, which eventually promotes Notch1 to rescue blockade in CD25+CD44+c-Kit+DN2 to CD25+CD44-c-Kit-DN3 transition in T cell maturation and suppress Ikaros/IRF8/Pu.1 to prevent DN2-T to DC differentiation in tumor hosts. The CD5intTCRαßhigh DP3 population was also increased to endorse CD8+ T cell generation. Conclusion: NLGP rescues tumor-induced altered thymic events to generate more effector T cells to restrain tumor.

Tumor Arrests DN2 to DN3 Pro T Cell Transition and Promotes Its Conversion to Thymic Dendritic Cells by Reciprocally Regulating Notch1 and Ikaros Signaling.

Tumor progression in the host leads to severe impairment of intrathymic T-cell differentiation/maturation, leading to the paralysis of cellular anti-tumor immunity. Such suppression manifests the erosion of CD4+CD8+ double-positive (DP) immature thymocytes and a gradual increase in CD4-CD8- double negative (DN) early T-cell progenitors. The impact of such changes on the T-cell progenitor pool in the context of cancer remains poorly investigated. Here, we show that tumor progression blocks the transition of Lin-Thy1.2+CD25+CD44+c-KitlowDN2b to Lin-Thy1.2+CD25+CD44-c-Kit-DN3 in T-cell maturation, instead leading to DN2-T-cell differentiation into dendritic cells (DC). We observed that thymic IL-10 expression is upregulated, particularly at cortico-medullary junctions (CMJ), under conditions of progressive disease, resulting in the termination of IL-10Rhigh DN2-T-cell maturation due to dysregulated expression of Notch1 and its target, CCR7 (thus restricting these cells to the CMJ). Intrathymic differentiation of T-cell precursors in IL-10-/- mice and in vitro fetal thymic organ cultures revealed that IL-10 promotes the interaction between thymic stromal cells and Notch1low DN2-T cells, thus facilitating these DN2-T cells to differentiate toward CD45+CD11c+MHC-II+ thymic DCs as a consequence of activating the Ikaros/IRF8 signaling axis. We conclude that a novel function of thymically-expressed IL-10 in the tumor-bearing host diverts T-cell differentiation toward a DC pathway, thus limiting the protective adaptive immune repertoire.

NLGP Attenuates Murine Melanoma and Carcinoma Metastasis by Modulating Cytotoxic CD8+ T Cells.

Neem leaf glycoprotein (NLGP), a natural immunomodulator, attenuates murine carcinoma and melanoma metastasis, independent of primary tumor growth and alterations in basic cellular properties (cell proliferation, cytokine secretion, etc.). Colonization event of invasion-metastasis cascade was primarily inhibited by NLGP, with no effect on metastasis-related invasion, migration, and extravasation. High infiltration of interferon γ (IFN-γ)-secreting cytotoxic CD8+ T cells [CD44+, CD69+, GranB+, IFN-γ+, and interleukin 2+] was documented in the metastatic site of NLGP-treated mice. Systemic CD8+ T cell depletion abolished NLGP-mediated metastasis inhibition and reappeared upon adoptive transfer of NLGP-activated CD8+ T cells. Interferon γ-secreting from CD8+ T cells inhibit the expression of angiogenesis regulatory vascular endothelial growth factor and transforming growth factor ß and have an impact on the prevention of colonization. Neem leaf glycoprotein modulates dendritic cells (DCs) for proper antigen presentation by its DC surface binding and upregulation of MHC-I/II, CD86, and CCR7. Neem leaf glycoprotein-treated DCs specifically imprint CXCR3 and CCR4 homing receptors on activated CD8+ T cells, which helps to infiltrate into metastatic sites to restrain colonization. Such NLGP's effect on DCs is translation dependent and transcription independent. Studies using ovalbumin, OVA257-264, and crude B16F10 antigen indicate MHC-I upregulation depends on the quantity of proteasome degradable peptide and only stimulates CD8+ T cells in the presence of antigen. Overall data suggest NLGP inhibits metastasis, in conjunction with tumor growth restriction, and thus might appear as a promising next-generation cancer immunotherapeutic.

Neem Leaf Glycoprotein Restrains VEGF Production by Direct Modulation of HIF1α-Linked Upstream and Downstream Cascades.

Neem Leaf Glycoprotein (NLGP) is a natural immunomodulator, have shown sustained tumor growth restriction as well as angiogenic normalization chiefly by activating CD8+ T cells. Here, we have investigated the direct role of NLGP as a regulator of tumor microenvironmental hypoxia and associated vascular endothelial growth factor (VEGF) production. We observed a significant reduction in VEGF level in both in vivo murine tumor and in vitro cancer cells (B16Mel, LLC) and macrophages after NLGP treatment. Interestingly, NLGP mediated VEGF downregulation in tumor cells or macrophages within hypoxic chamber was found at an early 4 h and again at late 24 h in mRNA level. Our data suggested that NLGP prevented hypoxia-induced strong binding of HIF1α with its co-factors, CBP/p300 and Sp3, but not with Sp1, which eventually limit the binding of HIF1α-transcriptional complex to hypoxia responsive element of VEGF promoter and results in restricted early VEGF transcription. On the otherhand, suppressed phosphorylation of Stat3 by NLGP results reduction of HIF1α at 24 h of hypoxia that further support sustained VEGF down-regulation. However, NLGP fails to regulate VHL activity as observed by both in vivo and in vitro studies. Therefore, this study for the first time reveals a mechanistic insight of NLGP mediated inhibition of angiogenesis by suppressing VEGF, which might help in vascular normalization to influence better drug delivery.