Hit-to-lead optimization of 2-aminoquinazolines as anti-microbial agents against Leishmania donovani.

Visceral leishmaniasis is a potentially fatal disease caused by infection by the intracellular protist pathogens Leishmania donovani or Leishmania infantum. Present therapies are ineffective because of high costs, variable efficacy against different species, the requirement for hospitalization, toxicity and drug resistance. Detailed analysis of previously published hit molecules suggested a crucial role of 'guanidine' linkage for their efficacy against L. donovani. Here we report the design of 2-aminoquinazoline heterocycle as a basic pharmacophore-bearing guanidine linkage. The introduction of various groups and functionality at different positions of the quinazoline scaffold results in enhanced antiparasitic potency with modest host cell cytotoxicity using a physiologically relevant THP-1 transformed macrophage infection model. In terms of the ADME profile, the C7 position of quinazoline was identified as a guiding tool for designing better molecules. The good ADME profile of the compounds suggests that they merit further consideration as lead compounds for treating visceral leishmaniasis.

Antimicrobial Activity of Trigonelline Hydrochloride Against Pseudomonas aeruginosa and Its Quorum-Sensing Regulated Molecular Mechanisms on Biofilm Formation and Virulence.

Pseudomonas aeruginosa, a vivid biofilm-producing bacterium, is considered a dreadful opportunistic pathogen, and thus, management of biofilm-associated infections due to multidrug resistant strains by traditional drugs currently is of great concern. This study was aimed to assess the impact of trigonelline hydrochloride, a pyridine alkaloid, on P. aeruginosa PAO1, in search of an alternative therapeutant. The effect of trigonelline on colony morphology and motility was studied along with its role on biofilm and expression virulence factors. Trigonelline influenced the colony structure, motility, biofilm architecture, and the production of virulence factors in a dose-dependent manner. Alterations in quorum sending (QS)-regulated gene expression after treatment and molecular docking analysis for certain regulator proteins confirmed its effect on the QS-system network by affecting Las, Rhl, and Pqs signaling pathways and as possible molecular targets. Thus, trigonelline might be considered as a potential chemical lead to manage biofilm-associated pathogenesis or to develop other analogues with enhanced pharmacokinetic actions.

The HBV web: An insight into molecular interactomes between the hepatitis B virus and its host en route to hepatocellular carcinoma.

Hepatitis B virus (HBV) is a major aetiology associated with the development and progression of hepatocellular carcinoma (HCC), the most common primary liver malignancy. Over the past few decades, direct and indirect mechanisms have been identified in the pathogenesis of HBV-associated HCC which include altered signaling pathways, genome integration, mutation-induced genomic instability, chromosomal deletions and rearrangements. Intertwining of the HBV counterparts with the host cellular factors, though well established, needs to be systemized to understand the dynamics of host-HBV crosstalk and its consequences on HCC progression. Existence of a vast array of protein-protein and protein-nucleic acid interaction databases has led to the uncoiling of the compendia of genes/gene products associated with these interactions. This review covers the existing knowledge about the HBV-host interplay and brings it down under one canopy emphasizing on the HBV-host interactomics; and thereby highlights new strategies for therapeutic advancements against HBV-induced HCC.

In Silico Integration of Transcriptome and Interactome Predicts an ETP-ALL-Specific Transcriptional Footprint that Decodes its Developmental Propensity.

Early T precursor acute lymphoblastic leukemia (ETP-ALL) exhibits poor clinical outcomes and high relapse rates following conventional chemotherapeutic protocols. Extensive developmental flexibility of the multipotent ETP-ALL blasts with considerable intra-population heterogeneity in terms of immunophenotype and prognostic parameters might be a target for novel therapeutic interventions. Using a public gene expression dataset (GSE28703) from NCBI GEO DataSets with 12 ETP-ALL and 40 non-ETP-ALL samples, such heterogeneity was found to be reflected in their transcriptome as well. Hub genes were identified from the STRING-derived functional interaction network of genes showing differential expression between ETP-ALL and non-ETP-ALL as well as variable expression across ETP-ALL. Nine genes (KIT, HGF, NT5E, PROM1, CD33, ANPEP, CDH2, IL1B, and CXCL2) among the hubs were further validated as possible diagnostic ETP-ALL markers using another gene expression dataset (GSE78132) with 17 ETP-ALL and 27 non-ETP-ALL samples. Linear dimensionality reduction analysis with the expression levels of the hub genes in ETP-ALL revealed their divergent inclinations towards different hematopoietic lineages, proposing them as novel indicators of lineage specification in the incompletely differentiated ETP-ALL blasts. This further led to the formulation of a personalized lineage score calculation algorithm, which uncovered a considerable B-lineage-bias in a substantial fraction of ETP-ALL subjects from the GSE28703 and GSE78132 cohorts. In addition, STRING-derived physical interactome of the potential biomarkers displayed complete segregation of the B-lineage-skewed markers from other lineage-associated factors, highlighting their distinct functionality and possible druggability in ETP-ALL. A panel of these biomarkers might be useful in pinpointing the dominant lineage specification programmes in the ETP-ALL blasts on a personalized level, urging the development of novel lineage-directed precision therapies as well as repurposing of existing therapies against leukemia of different hematopoietic lineages; which might overcome the drawbacks of conventional chemotherapy.

Familiarity Breeds Strategy: In Silico Untangling of the Molecular Complexity on Course of Autoimmune Liver Disease-to-Hepatocellular Carcinoma Transition Predicts Novel Transcriptional Signatures.

Autoimmune liver diseases (AILD) often lead to transformation of the liver tissues into hepatocellular carcinoma (HCC). Considering the drawbacks of surgical procedures in such cases, need of successful non-invasive therapeutic strategies and treatment modalities for AILD-associated-HCC still exists. Due to the lack of clear, sufficient knowledge about factors mediating AILD-to-HCC transition, an in silico approach was adopted to delineate the underlying molecular deterministic factors. Parallel enrichment analyses on two different public microarray datasets (GSE159676 and GSE62232) pinpointed the core transcriptional regulators as key players. Correlation between the expression kinetics of these transcriptional modules in AILD and HCC was found to be positive primarily with the advancement of hepatic fibrosis. Most of the regulatory interactions were operative during early (F0-F1) and intermediate fibrotic stages (F2-F3), while the extent of activity in the regulatory network considerably diminished at late stage of fibrosis/cirrhosis (F4). Additionally, most of the transcriptional targets with higher degrees of connectivity in the regulatory network (namely DCAF11, PKM2, DGAT2 and BCAT1) may be considered as potential candidates for biomarkers or clinical targets compared to their low-connectivity counterparts. In summary, this study uncovers new possibilities in the designing of novel prognostic and therapeutic regimen for autoimmunity-associated malignancy of liver in a disease progression-dependent fashion.

Type II Cytokines Fine-Tune Thymic T Cell Selection to Offset Murine Central Nervous System Autoimmunity.

Early thymic progenitors (ETPs) are bone marrow-derived hematopoietic stem cells that remain multipotent and give rise to a variety of lineage-specific cells. Recently, we discovered a subset of murine ETPs that expresses the IL-4Rα/IL-13Rα1 heteroreceptor (HR) and commits only to the myeloid lineage. This is because IL-4/IL-13 signaling through the HR inhibits their T cell potential and enacts commitment of HR+ETPs to thymic resident CD11c+CD8α+ dendritic cells (DCs). In this study, we discovered that HR+-ETP-derived DCs function as APCs in the thymus and promote deletion of myelin-reactive T cells. Furthermore, this negative T cell selection function of HR+-ETP-derived DCs sustains protection against experimental allergic encephalomyelitis, a mouse model for human multiple sclerosis. These findings, while shedding light on the intricacies underlying ETP lineage commitment, reveal a novel, to our knowledge, function by which IL-4 and IL-13 cytokines condition thymic microenvironment to rheostat T cell selection and fine-tune central tolerance.

A New IRF-1-Driven Apoptotic Pathway Triggered by IL-4/IL-13 Kills Neonatal Th1 Cells and Weakens Protection against Viral Infection.

Early life immune responses are deficient in Th1 lymphocytes that compromise neonatal vaccination. We found that IL-4 and IL-13 engage a developmentally expressed IL-4Rα/IL-13Rα1 heteroreceptor to endow IFN regulatory factor 1 (IRF-1) with apoptotic functions, which redirect murine neonatal Th1 reactivation to cell death. IL-4/IL-13-induced STAT6 phosphorylation serves to enhance IRF-1 transcription and promotes its egress from the nucleus. In the cytoplasm, IRF-1 can no longer serve as an anti-viral transcription factor but, instead, colocalizes with Bim and instigates the mitochondrial, or intrinsic, death pathway. The new pivotal function of IRF-1 in the death of neonatal Th1 cells stems from the ability of its gene to bind STAT6 for enhanced transcription and the proficiency of its protein to precipitate Bim-driven apoptosis. This cytokine-induced, IRF-1-mediated developmental death network weakens neonatal Th1 responses during early life vaccination and increases susceptibility to viral infection.

IL-4 and IL-13 Guide Early Thymic Progenitors To Mature toward Dendritic Cells.

Recently we reported that IL-4 and IL-13 signaling in murine early thymic progenitors (ETPs) expressing the heteroreceptor (HR) comprising IL-4 receptor α (IL-4Rα) and IL-13 receptor α 1 (IL-13Rα1) activate STAT6 and inhibit ETP maturation potential toward T cells. In this study, we asked whether IL-4 and IL-13 signaling through the HR mobilizes other STAT molecules to shape ETP fate decision. The findings indicate that HR+ ETPs undergoing cytokine signaling display increased STAT1, but not STAT3, phosphorylation in addition to STAT6 activation. In parallel, the ETPs had a STAT1-dependent heightened expression of IRF-8, a transcription factor essential for development of CD8α+ dendritic cells (DCs). Interestingly, STAT1 phosphorylation and IRF-8 upregulation, which are independent of STAT6 activation, guided ETP maturation toward myeloid cells with a CD8α+ DC phenotype. Furthermore, these CD8α+ DCs display a thymic resident phenotype, as they did not express SIRPα, a molecule presumed to be involved in cell migration. These findings suggest that IL-4 and IL-13 cytokine-induced HR signaling provides a double-edged sword that simultaneously blocks T cell lineage potential but advances myeloid maturation that could impact T cell selection and central tolerance.

A distinct dendritic cell population arises in the thymus of IL-13Rα1-sufficient but not IL-13Rα1-deficient mice.

IL-13 receptor alpha 1 (IL-13Rα1) associates with IL-4Rα to form a functional IL-4Rα/IL-13Rα1 heteroreceptor (HR) through which both IL-4 and IL-13 signal. Recently, HR expression was associated with the development of M2 type macrophages which function as antigen presenting cells (APCs). Herein, we show that a subset of thymic resident dendritic cells (DCs) expressing high CD11b (CD11bhi) and intermediate CD11c (CD11cint) arise in HR-sufficient but not HR-deficient mice. These DCs, which originate from the bone marrow are able to take up Ag from the peritoneum, traffic through the spleen and the lymph nodes and carry it to the thymus. In addition, since the DCs are able to present Ag to T cells, express high levels of the costimulatory molecule CD24, and comprise a CD8α+ subset, it is likely that the cells contribute to T cell development and perhaps negative selection of self-reactive lymphocytes.

IL-4/IL-13 Signaling Inhibits the Potential of Early Thymic Progenitors To Commit to the T Cell Lineage.

Early thymic progenitors (ETPs) are endowed with diverse potencies and can give rise to myeloid and lymphoid lineage progenitors. How the thymic environment guides ETP commitment and maturation toward a specific lineage remains obscure. We have previously shown that ETPs expressing the heteroreceptor (HR) comprising IL-4Rα and IL-13Rα1 give rise to myeloid cells but not T cells. In this article, we show that signaling through the HR inhibits ETP maturation to the T cell lineage but enacts commitment toward the myeloid cells. Indeed, HR+ ETPs, but not HR- ETPs, exhibit activated STAT6 transcription factor, which parallels with downregulation of Notch1, a critical factor for T cell development. Meanwhile, the myeloid-specific transcription factor C/EBPα, usually under the control of Notch1, is upregulated. Furthermore, in vivo inhibition of STAT6 phosphorylation restores Notch1 expression in HR+ ETPs, which regain T lineage potential. In addition, upon stimulation with IL-4 or IL-13, HR- ETPs expressing virally transduced HR also exhibit STAT6 phosphorylation and downregulation of Notch1, leading to inhibition of lymphoid, but not myeloid, lineage potential. These observations indicate that environmental cytokines play a role in conditioning ETP lineage choice, which would impact T cell development.

IL-4/IL-13 Heteroreceptor Influences Th17 Cell Conversion and Sensitivity to Regulatory T Cell Suppression To Restrain Experimental Allergic Encephalomyelitis.

IL-4 and IL-13 have been defined as anti-inflammatory cytokines that can counter myelin-reactive T cells and modulate experimental allergic encephalomyelitis. However, it is not known whether endogenous IL-4 and IL-13 contribute to the maintenance of peripheral tolerance and whether their function is coordinated with T regulatory cells (Tregs). In this study, we used mice in which the common cytokine receptor for IL-4 and IL-13, namely the IL-4Rα/IL-13Rα1 (13R) heteroreceptor (HR), is compromised and determined whether the lack of signaling by endogenous IL-4 and IL-13 through the HR influences the function of effector Th1 and Th17 cells in a Treg-dependent fashion. The findings indicate that mice-deficient for the HR (13R-/-) are more susceptible to experimental allergic encephalomyelitis than mice sufficient for the HR (13R+/+) and develop early onset and more severe disease. Moreover, Th17 cells from 13R-/- mice had reduced ability to convert to Th1 cells and displayed reduced sensitivity to suppression by Tregs relative to Th17 effectors from 13R+/+ mice. These observations suggest that IL-4 and IL-13 likely operate through the HR and influence Th17 cells to convert to Th1 cells and to acquire increased sensitivity to suppression, leading to control of immune-mediated CNS inflammation. These previously unrecognized findings shed light on the intricacies underlying the contribution of cytokines to peripheral tolerance and control of autoimmunity.

Neem leaf glycoprotein generates superior tumor specific central memory CD8+ T cells than cyclophosphamide that averts post-surgery solid sarcoma recurrence.

The success of cancer vaccines is limited as most of them induce corrupted CD8+ T cell memory populations. We reported earlier that a natural immunomodulator, neem leaf glycoprotein (NLGP), therapeutically restricts tumor growth in a CD8+ T cell-dependent manner. Here, our objective is to study whether memory CD8+ T cell population is generated in sarcoma hosts after therapeutic NLGP treatment and their role in prevention of post-surgery tumor recurrence, in comparison to the immunostimulatory metronomic cyclophosphamide (CTX) treatment. We found that therapeutic NLGP and CTX treatment generates central memory CD8+ T (TCM) cells with characteristic CD44+CD62LhighCCR7highIL-2high phenotypes. But these TCM cells are functionally impaired to prevent re-appearance of tumors along with compromised proliferative, IL-2 secretive and cytotoxic status. This might be due to the presence of tumor load, even a small one in the host, which serves as a persistent source of tumor antigens thereby corrupting the TCM cells so generated. Surgical removal of the persisting tumors from the host restored the functional characteristics of memory CD8+ T cells, preventing tumor recurrence after surgery till end of the experiment. Moreover, we observed that generation of superior TCM cells in NLGP treated surgically removed tumor hosts is related to the activation of Wnt signalling in memory CD8+ T cells with concomitant inhibition of GSK-3ß and stabilisation of ß-catenin, which ultimately activates transcription of Wnt target genes, like, eomesodermin, a signature molecule of CD8+ TCM cells.

On the Role IL-4/IL-13 Heteroreceptor Plays in Regulation of Type 1 Diabetes.

Type 1 diabetes (T1D) manifests when the insulin-producing pancreatic ß cells are destroyed as a consequence of an inflammatory process initiated by lymphocytes of the immune system. The NOD mouse develops T1D spontaneously and serves as an animal model for human T1D. The IL-4Rα/IL-13Rα1 heteroreceptor (HR) serves both IL-4 and IL-13 cytokines, which are believed to function as anti-inflammatory cytokines in T1D. However, whether the HR provides a responsive element to environmental (i.e., physiologic) IL-4/IL-13 in the regulation of peripheral tolerance and the development of T1D has yet to be defined. In this study, NOD mice deficient for the HR have been generated by means of IL-13Rα1 gene disruption and used to determine whether such deficiency affects the development of T1D. Surprisingly, the findings indicate that NOD mice lacking the HR (13R-/-) display resistance to T1D as the rise in blood glucose level and islet inflammation were significantly delayed in these HR-deficient relative to HR-sufficient (13R+/+) mice. In fact, the frequency and spleen-to-pancreas dynamics of both Th1 and Th17 cells were affected in 13R-/- mice. This is likely due to an increase in the frequency of mTGFß+Foxp3int regulatory T cells and the persistence of CD206+ macrophages in the pancreas as both types of cells confer resistance to T1D upon transfer to 13R+/+ mice. These findings reveal new insights as to the role environmental IL-4/IL-13 and the HR play in peripheral tolerance and the development of T1D.

Neem leaf glycoprotein prevents post-surgical sarcoma recurrence in Swiss mice by differentially regulating cytotoxic T and myeloid-derived suppressor cells.

Post-surgical tumor recurrence is a common problem in cancer treatment. In the present study, the role of neem leaf glycoprotein (NLGP), a novel immunomodulator, in prevention of post-surgical recurrence of solid sarcoma was examined. Data suggest that NLGP prevents tumor recurrence after surgical removal of sarcoma in Swiss mice and increases their tumor-free survival time. In NLGP-treated tumor-free mice, increased cytotoxic CD8+ T cells and a decreased population of suppressor cells, especially myeloid-derived suppressor cells (MDSCs) was observed. NLGP-treated CD8+ T cells showed greater cytotoxicity towards tumor-derived MDSCs and supernatants from the same CD8+ T cell culture caused upregulation of FasR and downregulation of cFLIP in MDSCs. To elucidate the role of CD8+ T cells, specifically in association with the downregulation in MDSCs, CD8+ T cells were depleted in vivo before NLGP immunization in surgically tumor removed mice and tumor recurrence was noted. These mice also exhibited increased MDSCs along with decreased levels of Caspase 3, Caspase 8 and increased cFLIP expression. In conclusion, it can be stated that NLGP, by activating CD8+ T cells, down regulates the proportion of MDSCs. Accordingly, suppressive effects of MDSCs on CD8+ T cells are minimized and optimum immune surveillance in tumor hosts is maintained to eliminate the residual tumor mass appearing during recurrence.

Neem leaf glycoprotein regulates function of tumor associated M2 macrophages in hypoxic tumor core: Critical role of IL-10/STAT3 signaling.

Heterogeneous tumor microenvironment (TME), broadly divided into tumor core and peripheral sub-microenvironments, differentially polarize normal macrophages into a different form known as tumor associated M2 macrophages (M2TAMs) to promote tumor growth. In view of the extensive immune-editing role of NLGP, here, we have observed that NLGP is effective to convert M2TAMs (CD11b+F4/80high) to M1 (CD11b+F4/80low) more prominently in tumor core, along with downregulation of other M2 associated markers, like, ManR, Ym1, Fizz1. High IL-10:IL-12 ratio at tumor core was downregulated in NLGP treated melanoma bearing mice. Decrease in IL-10 by NLGP is again associated with the decrease in hypoxia, as indicated by prominent downregulation of HIF1α and VEGF, particularly at tumor core. Macrophages exposed to hypoxic tumor core lysates in vitro exhibited high IL-10, HIF1α and VEGF expression that was significantly downregulated by NLGP. Further evidences suggest M2TAM to M1 conversion by NLGP is associated with STAT3-regulated IL-10 dependent pathway without affecting the IL-4 dependent one. Such TAM modulatory functions of NLGP might help in the restriction of melanoma growth by increasing the proportion of M1 TAMs in tumor core that helps in prevention of tumor relapse and dissemination of the tumor mass.

Antigen-Specific Immune Modulation Targets mTORC1 Function To Drive Chemokine Receptor-Mediated T Cell Tolerance.

To contain autoimmunity, pathogenic T cells must be eliminated or diverted from reaching the target organ. Recently, we defined a novel form of T cell tolerance whereby treatment with Ag downregulates expression of the chemokine receptor CXCR3 and prevents diabetogenic Th1 cells from reaching the pancreas, leading to suppression of type 1 diabetes (T1D). This report defines the signaling events underlying Ag-induced chemokine receptor-mediated tolerance. Specifically, we show that the mammalian target of rapamycin complex 1 (mTORC1) is a major target for induction of CXCR3 downregulation and crippling of Th1 cells. Indeed, Ag administration induces upregulation of programmed death-ligand 1 on dendritic cells in a T cell-dependent manner. In return, programmed death-ligand 1 interacts with the constitutively expressed programmed death-1 on the target T cells and stimulates docking of Src homology 2 domain-containing tyrosine phosphatase 2 phosphatase to the cytoplasmic tail of programmed death-1. Active Src homology 2 domain-containing tyrosine phosphatase 2 impairs the signaling function of the PI3K/protein kinase B (AKT) pathway, leading to functional defect of mTORC1, downregulation of CXCR3 expression, and suppression of T1D. Thus, mTORC1 component of the metabolic pathway serves as a target for chemokine receptor-mediated T cell tolerance and suppression of T1D.

Tumor-associated mesenchymal stem cells inhibit naïve T cell expansion by blocking cysteine export from dendritic cells.

Mesenchymal stem cells (MSCs) represent an important cellular constituent of the tumor microenvironment, which along with tumor cells themselves, serve to regulate protective immune responses in support of progressive disease. We report that tumor MSCs prevent the ability of dendritic cells (DC) to promote naïve CD4(+) and CD8(+) T cell expansion, interferon gamma secretion and cytotoxicity against tumor cells, which are critical to immune-mediated tumor eradication. Notably, tumor MSCs fail to prevent DC-mediated early T cell activation events or the ability of responder T cells to produce IL-2. The immunoregulatory activity of tumor MSCs is IL-10- and STAT3-dependent, with STAT3 repressing DC expression of cystathionase, a critical enzyme that converts methionine-to-cysteine. Under cysteine-deficient priming conditions, naïve T cells exhibit defective cellular metabolism and proliferation. Bioinformatics analyses as well as in vitro observations suggest that STAT3 may directly bind to a GAS-like motif within the cystathionase promoter (-269 to -261) leading to IL-10-STAT3 mediated repression of cystathionase gene transcription. Our collective results provide evidence for a novel mechanism of tumor MSC-mediated T cell inhibition within tumor microenvironment.

Neem leaf glycoprotein promotes dual generation of central and effector memory CD8(+) T cells against sarcoma antigen vaccine to induce protective anti-tumor immunity.

We have previously shown that Neem Leaf Glycoprotein (NLGP) mediates sustained tumor protection by activating host immune response. Now we report that adjuvant help from NLGP predominantly generates CD44(+)CD62L(high)CCR7(high) central memory (TCM; in lymph node) and CD44(+)CD62L(low)CCR7(low) effector memory (TEM; in spleen) CD8(+) T cells of Swiss mice after vaccination with sarcoma antigen (SarAg). Generated TCM and TEM participated either to replenish memory cell pool for sustained disease free states or in rapid tumor eradication respectively. TCM generated after SarAg+NLGP vaccination underwent significant proliferation and IL-2 secretion following SarAg re-stimulation. Furthermore, SarAg+NLGP vaccination helps in greater survival of the memory precursor effector cells at the peak of the effector response and their maintenance as mature memory cells, in comparison to single modality treatment. Such response is corroborated with the reduced phosphorylation of FOXO in the cytosol and increased KLF2 in the nucleus associated with enhanced CD62L, CCR7 expression of lymph node-resident CD8(+) T cells. However, spleen-resident CD8(+) T memory cells show superior efficacy for immediate memory-to-effector cell conversion. The data support in all aspects that SarAg+NLGP demonstrate superiority than SarAg vaccination alone that benefits the host by rapid effector functions whenever required, whereas, central-memory cells are thought to replenish the memory cell pool for ultimate sustained disease free survival till 60 days following post-vaccination tumor inoculation.

Neem leaf glycoprotein prophylaxis transduces immune dependent stop signal for tumor angiogenic switch within tumor microenvironment.

We have reported that prophylactic as well as therapeutic administration of neem leaf glycoprotein (NLGP) induces significant restriction of solid tumor growth in mice. Here, we investigate whether the effect of such pretreatment (25µg/mice; weekly, 4 times) benefits regulation of tumor angiogenesis, an obligate factor for tumor progression. We show that NLGP pretreatment results in vascular normalization in melanoma and carcinoma bearing mice along with downregulation of CD31, VEGF and VEGFR2. NLGP pretreatment facilitates profound infiltration of CD8+ T cells within tumor parenchyma, which subsequently regulates VEGF-VEGFR2 signaling in CD31+ vascular endothelial cells to prevent aberrant neovascularization. Pericyte stabilization, VEGF dependent inhibition of VEC proliferation and subsequent vascular normalization are also experienced. Studies in immune compromised mice confirmed that these vascular and intratumoral changes in angiogenic profile are dependent upon active adoptive immunity particularly those mediated by CD8+ T cells. Accumulated evidences suggest that NLGP regulated immunomodulation is active in tumor growth restriction and normalization of tumor angiogenesis as well, thereby, signifying its clinical translation.

Immunotherapeutic targeting of established sarcoma in Swiss mice by tumor-derived antigen-pulsed NLGP matured dendritic cells is CD8+ T-cell dependent.

AIM: Neem leaf glycoprotein (NLGP) matures human myeloid and mouse bone marrow-derived dendritic cells (DCs). (NLGP) also therapeutically restricts the mouse established sarcoma growth by activating CD8(+) T cells along with increased proportion of tumor residing CD11c(+) DCs. Here, we intended to find out whether CD8(+) T cells become cytotoxic to sarcoma cells after presentation of sarcoma antigen by NLGP-matured DCs to restrict murine sarcoma growth. MATERIALS & METHODS: NLGP was prepared from matured neem(Azadirachta indica) leaves. Solid sarcoma tumor in Swiss mice was developed by subcutaneous inoculation of sarcoma cells. GMCSF-IL-4 generated DCs were matured with NLGP and pulsed with sarcoma antigen for immunotherapy. Status of CD8+CD69+T cells was studied by flow cytometry and secretion of cytokines was measured by ELISA. RT-PCR was used to monitor the status of perforin, granzyme B. RESULTS: NLGP-matured sarcoma antigen-pulsed DCs (DCNLGPTAg) inhibit mouse sarcoma growth. DCNLGPTAg immunization enhances CD8(+) T-cell number within tumor-infiltrating lymphocytes and tumor-draining lymph nodes along with increased perforin and granzyme B expression. Antigen-specific T-cell proliferation and IFN-γ secretion were significantly higher in DCNLGP- and DCNLGPTAg-immunized mice groups. In vivo CD8(+) T-cell depletion abrogated the DCNLGPTAg-mediated tumor growth restriction. CONCLUSION: DCNLGPTAg restricts CD8(+) T-cell-dependent mouse established sarcoma growth, related to the optimum antigen presentation by DCs to CD8(+) T cells.