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 β-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 β-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.
CONCLUSIONS: 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.

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.
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.
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.
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.

A dynamic interaction between tumor cells and its surrounding stroma promotes the initiation, progression, metastasis, and chemoresistance of solid tumors. Emerging evidences suggest that targeting the stromal events could improve the efficacies of current therapeutics. Within tumor microenvironment (TME), stromal progenitor cells, i.e., MSCs, interact and eventually modulate the biology and functions of cancer and immune cells. Our recent finding disclosed a novel mechanism stating that tumor-associated MSCs inhibit the T cell proliferation and effector functions by blocking cysteine transport to T cells by dendritic cells (DCs), which makes MSCs as a compelling candidate as a therapeutic target. Immunomodulation by nontoxic neem leaf glycoprotein (NLGP) on dysfunctional cancer immunity offers significant therapeutic benefits to murine tumor host; however, its modulation on MSCs and its impact on T cell functions need to be elucidated.
Bone marrow-derived primary MSCs or murine 10 T1/2 MSCs were tumor-conditioned (TC-MSCs) and co-cultured with B16 melanoma antigen-specific DCs and MACS purified CD4+ and CD8+ T cells. T cell proliferation of T cells was checked by Ki67-based flow-cytometric and thymidine-incorporation assays. Cytokine secretion was measured by ELISA. The expression of cystathionase in DCs was assessed by RT-PCR. The STAT3/pSTAT3 levels in DCs were assessed by western blot, and STAT3 function was confirmed using specific SiRNA. Solid B16 melanoma tumor growth was monitored following adoptive transfer of conditioned CD8+ T cells.
NLGP possesses an ability to restore anti-tumor T cell functions by modulating TC-MSCs. Supplementation of NLGP in DC-T cell co-culture significantly restored the inhibition in T cell proliferation and IFNγ secretion almost towards normal in the presence of TC-MSCs. Adoptive transfer of NLGP-treated TC-MSC supernatant educated CD8+ T cells in solid B16 melanoma bearing mice resulted in better tumor growth restriction than TC-MSC conditioned CD8+ T cells. NLGP downregulates IL-10 secretion by TC-MSCs, and concomitantly, pSTAT3 expression was downregulated in DCs in the presence of NLGP-treated TC-MSC supernatant. As pSTAT3 negatively regulates cystathionase expression in DCs, NLGP indirectly helps to maintain an almost normal level of cystathionase gene expression in DCs making them able to export sufficient amount of cysteine required for optimum T cell proliferation and effector functions within TME.
NLGP could be a prospective immunotherapeutic agent to control the functions and behavior of highly immunosuppressive TC-MSCs providing optimum CD8+ T cell functions to showcase an important new approach that might be effective in overall cancer treatment.

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-3β and stabilisation of β-catenin, which ultimately activates transcription of Wnt target genes, like, eomesodermin, a signature molecule of CD8+ TCM cells.

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.

One of the prime objectives of cancer immunology and immunotherapy is to study the issues related to rescue and/or maintenance of the optimum effector CD8(+) T cell functions by minimizing tumor-induced negative factors. In this regard the influence of host intrinsic CD4(+) helper T cells towards generation and maintenance of CD8(+) effector T cells appears controversial in different experimental settings. Therefore, the present study was aimed to re-analyze the influence of CD4(+) helper T cells towards effector T cells during neem leaf glycoprotein (NLGP)-vaccine-mediated tumor growth restriction. CD4 depletion (mAb; Clone GK1.5) surprisingly resulted in significant increase in CD8(+) T cells in different immune organs from NLGP-treated sarcoma-bearing mice. However, such CD8 surge could not restrict the sarcoma growth in NLGP-treated CD4-depleted mice. Furthermore, CD4 depletion in early phase hinders CD8(+) T cell activation and terminal differentiation by targeting crucial transcription factor Runx3. CD4 depletion decreases accumulation of CD8α(+) dendritic cells within tumor draining lymph node, hampers antigen cross priming and CD86-CD28 interactions for optimum CD8(+) T cell functions. In order to search the mechanism of CD4(+) T cell help on NLGP-mediated CD8 effector functions, the role of CD4(+) helper T cell-derived IL-2 on optimization of CD8 functions was found using STAT5 signaling, but complete response requires physical contact of CD4(+) helper T cells with its CD8 counterpart. In conclusion, it was found that CD4(+) T cell help is not required to generate CD8(+) T cells but was found to be an integral phenomenon in maintenance of its anti-tumor functions even in NLGP-vaccine-mediated sarcoma growth restriction.

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.

We have generated a polyclonal antibody against a novel immunomodulator, neem leaf glycoprotein (NLGP) that can react to a specific 47 kDa subunit of NLGP. Generated anti-NLGP antibody (primarily IgG2a) was tested for its anti-tumor activity in murine carcinoma (EC, CT-26), sarcoma (S180) and melanoma (B16Mel) tumor models. Surprisingly, tumor growth restriction was only observed in CT-26 carcinoma models, without any alteration in other tumor systems. Comparative examination of antigenicity between four different tumor models revealed high expression of CEA-like protein on the surface of CT-26 tumors. Subsequent examination of the cross-reactivity of anti-NLGP antibody with purified or cell bound CEA revealed prominent recognition of CEA by anti-NLGP antibody, as detected by ELISA, Western Blotting and immunohistochemistry. This recognition seems to be responsible for anti-tumor function of anti-NLGP antibody only on CEA-like protein expressing CT-26 tumor models, as confirmed by ADCC reaction in CEA(+) tumor systems where dependency to anti-NLGP antibody is equivalent to anti-CEA antibody. Obtained result with enormous therapeutic potential for CEA(+) tumors may be explained in view of the epitope spreading concept, however, further investigation is crucial.

Tumor-associated macrophages (TAMs) are preferentially M2 skewed and promote tumor growth, angiogenesis, invasion, and/or metastasis. In this study, we have analyzed the in vitro immunomodulatory potential of a non-toxic neem leaf glycoprotein (NLGP) in reprogramming Stage III supraglottic laryngeal tumor cell lysate (SLTCL) induced M2 TAMs to their classical anti-tumor shape (M1). Data generated from this study support that NLGP is effective in preventing the SLTCL induced generation (CD68(+)CD206(+)IL-10(high) to CD68(+)CD206(-)IL-10(low) TAMs) and functions (NO(low) to NO(high), MHC-I(low) to MHC-I(high), CD80(low) to CD80(high)) of pro-tumorous M2 macrophages, which in turn associated with sustained anti-tumor effector functions by promoting cytotoxic T cell activities and suppressing regulatory T cells. Furthermore, our data also suggest that NLGP prevents M2 skewness of TAMs by downregulating phosphorylation of targeted STAT3.

We have observed earlier that therapeutic treatment with neem leaf glycoprotein (NLGP) inhibits murine B16-melanoma growth in vivo and improves survivability of treated mice. Anti-tumor effect of NLGP is directly associated with enhanced CD8(+) T cell activity and downregulation of suppressive cellular functions. Objective of this present study is to know the efficacy of NLGP in comparison to two popular drugs, Cisplatin and Sunitinib malate (Sutent) in relation to the modulation of tumor microenvironment (TME). Analysis of cytokine milieu within TME revealed IL-10, TGFβ, IL-6 rich type 2 characters was significantly switched to type 1 microenvironment with dominance of IFNγ and IL-2 within NLGP-TME, which was not found in other cases; however Cisplatin-TME appeared better in type 2 to type 1 conversion than Sutent-TME as evidenced by RT-PCR, ELISA and immunohistochemical analysis. NLGP-TME educated CD8(+) T cells exhibited greater cytotoxicity to B16 Melanoma cells in vitro and these cells showed comparatively higher expression of cytotoxicity related molecules, perforin and granzyme B than Cisplatin-TME and Sutent-TME educated T cells. Adoptive transfer of NLGP-TME exposed T cells, but not PBS-TME exposed cells in mice, is able to significantly inhibit the growth of melanoma in vivo. Such tumor growth inhibition was in significantly lower extent when therapeutic CD8(+) T cells were exposed to either Cisplatin-TME or Sutent-TME or control-TME. Accumulated evidences strongly suggest that non toxic NLGP normalized TME allows T cells to perform optimally than other TMEs under study to inhibit the melanoma growth.