Neem leaf glycoprotein directs T-bet-associated type 1 immune commitment.

Neem leaf glycoprotein (NLGP)-mediated immune activation and associated immune polarization was studied. NLGP-induced activation is reflected in upregulation of early activation marker CD69 on lymphocytes, monocytes, and dendritic cells. Activation is also denoted by CD45RO enhancement, with a decrease in CD45RA phenotype and CD62L (L-selectin). NLGP-activated T cells secrete greater amount of signature T-helper (Th)1 cytokines interferon-gamma and a lower amount of the Th2 cytokine interleukin (IL)-4. Similar type 1 directiveness is also observed in antigen-presenting monocytes and dendritic cells by upregulation of IL-12, tumor necrosis factor -alpha and downregulation of IL-10. Creation of the type 1 microenvironment is also assisted by NLGP-induced downregulation of FoxP3(+) T-Reg cells. A type 1-specific transcription factor, T-bet, is upregulated in circulating immune cells after their stimulation with NLGP. In the creation of type 1 immune network, increased phosphorylation of STAT1 and STAT4 with decreased phosphorylation of STAT3 might have significance. We conclude that NLGP may be effective in maintaining normal immune homeostasis by upregulating type 1 response in immunosuppressed hosts, which may have significant role in the induction of host protective antitumor functions.

Dysregulation in immune functions is reflected in tumor cell cytotoxicity by peripheral blood mononuclear cells from head and neck squamous cell carcinoma patients.

We assessed the immunological status of stage III and IV head and neck squamous cell carcinoma (HNSCC) patients and age-matched healthy individuals. In HNSCC patients, the total leukocyte count was lower and the proliferating ability of PBMCs against phytohemagglutinin (PHA) was significantly downregulated. These cells showed lower expression of the early activation marker CD69. Within this PBMC population, the proportion of CD4+, CD8+ T cells, CD3- CD56+, CD16+ NK cells and CD3+ CD56+ NK-T cells was seriously downregulated. However, the proportion of CD4+ CD25+ Foxp3+ regulatory T cells having suppressor function was upregulated. Other immune cells, like CD14+ monocytes/macrophages and CD20+ B cells, were also fewer in number, although this difference was not statistically significant. Assessment of the cytokine secretory status of PBMCs revealed suppressed levels of Th1 cytokines (IFN-gamma, IL-12 and TNF-alpha) and elevated secretion of Th2 cytokines (IL-4 and IL-10) for HNSCC PBMCs whereas just the opposite was seen for PBMCs from healthy individuals. Dysregulation in the profile of immunocompetent cells and cytokine secretion was reflected in the suppressed cytotoxic function of HNSCC PBMCs, as tested on KB (oral cancer), MCF7 (breast cancer), COLO205 (colon cancer), Jurkat (T cell leukemia), K562 (erythroleukemia) and U937 (monocytic lymphoma) cell lines. The observed decreased cytotoxicity of HNSCC PBMCs may be due to the downregulated expression of cytotoxic molecules (perforin, granzymeB and FasL) in HNSCC PBMCs. Assessment of the extent of immune dysfunction might help design immunotherapeutic protocols by incorporating any agent having immunomodulatory function.

Neem leaf glycoprotein overcomes indoleamine 2,3 dioxygenase mediated tolerance in dendritic cells by attenuating hyperactive regulatory T cells in cervical cancer stage IIIB patients.

Tolerogenic dendritic cells (DCs) are a subset of DCs characterized by abundant indoleamine 2,3 dioxygenase (IDO) expressions. IDO may be co-operatively induced in DCs by regulatory T (Tregs) cells and various DC maturation agents. Tregs are markedly amplified in the physiological system of cancer patients, inducing over tolerance in DCs that leads to the hyper accumulation of immunosuppressive IDO in tumor microenvironment, thereby, hampering anti-tumor immunity. Consequently, a major focus of current immunotherapeutic strategies in cancer is to minimize IDO, which is possible by reducing Tregs and using various IDO inhibitors. Neem leaf glycoprotein (NLGP), a natural and nontoxic immunomodulator, demonstrated several unique immunoregulatory activities. Noteworthy activities of NLGP are to mature DCs and to inhibit Tregs. As Tregs are inducer of IDO in DCs and hyperactive Tregs is a hallmark of cancer, we anticipated that NLGP might abrogate IDO induction in DCs by inhibiting Tregs. Evidences are presented here that in a co-culture of DCs and Tregs isolated from cervical cancer stage IIIB (CaCx-IIIB) patients, NLGP does inhibit IDO induction in DCs by curtailing the over expression of Cytotoxic T-Lymphocyte Antigen 4 (CTLA4) on Tregs and concomitantly induces optimal DC maturation. In contrast, in the presence of LPS as maturation agent the DCs displays a tolerogenic profile. This finding suggests the reduction of tolerogenecity of DCs in CaCx-IIIB patients by reducing the IDO pool using NLGP. Accordingly, this study sheds more light on the diverse immunomodulatory repertoire of NLGP.

Neem leaf glycoprotein suppresses regulatory T cell mediated suppression of monocyte/macrophage functions.

We have shown that neem leaf glycoprotein (NLGP) inhibits the regulatory T cell (Tregs) induced suppression of tumoricidal functions of CD14(+)CD68(+) monocyte/macrophages (MO/Mφ) from human peripheral blood. Cytotoxic efficacy of MO/Mφ toward macrophage sensitive cells, U937, is decreased in presence of Tregs (induced), however, it was increased further by supplementation of NLGP in culture. Associated Treg mediated inhibition of perforin/granzyme B expression and nitric oxide release from MO/Mφ was normalized by NLGP. Altered status of signature cytokines, like, IL-12, IL-10, IL-6, TNFα from MO/Mφ under influence of Tregs is also rectified by NLGP. Tregs significantly enhanced the expression of altered marker, mannose receptor (CD206) on CD68(+) cells that was downregulated upon NLGP exposure. In addition to tumoricidal functions, antigen presenting ability of MO/Mφ is hampered by Treg induced downregulation of CD80, CD86 and HLA-ABC. NLGP upregulated these molecules in MO/Mφ even in the presence of Tregs. Treg mediated inhibition of MO/Mφ chemotaxis in contact dependent manner was also normalized partially by NLGP, where participation of CCR5 was documented. Overall results suggest that Treg influenced pro-tumor MO/Mφ functions are rectified in a significant extent by NLGP to create an anti-tumor immune environment.

Dysregulated CC receptor/ligand in monocytes/macrophages from tongue squamous cell carcinoma patients is partially rectified by interferon α-2b.

In an aim to rectify dysregulated CC chemokine receptor (CCR5)/ligand (RANTES, MIP-1α, MIP-1ß) status of monocytes/macrophages in tongue squamous cell carcinoma (TSCC; n = 12) patients, we have tested interferon α2b (IFNα2b), a novel immunomodulator with wide use in the management of several forms of cancer. IFNα2b can upregulate reduced CCR5 expression and increases the suppressed secretory status of its ligands, as evidenced from in vitro studies on monocytes/macrophages from the peripheral blood of TSCC patients as well as healthy individuals. Isolated monocytes of TSCC patients (n = 5) undergoing chemotherapeutic treatment along with IFNα2b immunotherapy demonstrated significant upregulation in CCR5 expression and secretion of corresponding ligands. These rectifications in receptor/ligand levels are reflected in improved CCR5-dependent migration of monocytes/macrophages after IFNα2b treatment. The rectified chemokine profile and cellular migration translate into better tumoricidal and antigen-presenting functions of these cells. Accordingly, enhanced T-cell-mediated tumor cell killing is demonstrated upon IFNα2b treatment. Translating dual benefits on monocyte/macrophage functions, IFNα2b may emerge as a potential form of immunotherapy for TSCC patients that may be combined with standard chemotherapy for better clinical outcome.

Defective dendritic cell generation from monocytes is a potential reason for poor therapeutic efficacy of interferon α2b (IFNα2b) in cervical cancer.

Despite being a pleiotropic cytokine, the therapeutic potential of interferon α2b (IFNα2b) is debatable. Thus, the need for identifying predictive marker(s) for patients who are most likely to benefit from the treatment is pivotal for avoiding the exposure of nonresponsive patients to the toxicity of the treatment. To account for the attenuated efficacy of the drug, we have verified its dendritic cell (DC) maturating ability from monocytes of cervical cancer stage IIIB (CaCx-IIIB) patients. First, we evaluated the status of monocytes from CaCx-IIIB and healthy women by conducting flow cytometric studies of various activation markers and a cytokine analysis by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Immature DCs were then generated from these monocytes and matured with low-dose IFNα2b (1500 units/mL). A functional and phenotypic comparative analysis of these matured DCs was performed by flow cytometric, proliferative, cytotoxic, and enzyme-linked immunosorbent assays. Our study shows that monocytes isolated from CaCx-IIIB are impaired, and in vitro maturation with IFNα2b did not significantly improve the functional repertoire of DCs generated from these monocytes in comparison with healthy controls. This impairment of monocytes might be a plausible reason for the attenuated efficacy of this drug alone in treating CaCx-IIIB patients, and this imbalance of immune parameters associated with the stage of malignancy might be considered an effective marker to design a proper therapeutic regimen.

Neem leaf glycoprotein inhibits CD4+CD25+Foxp3+ Tregs to restrict murine tumor growth.

BACKGROUND: The presence of Tregs in tumors is associated with compromised tumor-specific immune responses and has a clear negative impact on survival of cancer patients. Thus, downregulation of Tregs is considered as a promising cancer immunotherapeutic approach. We have reported previously that neem leaf glycoprotein (NLGP) prophylaxis restricts tumor growth in mice by immune activation. In continuation, here, involvement of NLGP in the modulation of Tregs in association with tumor growth restriction is investigated. RESULTS: NLGP downregulates CD4+CD25+Foxp3+ Tregs within tumors. NLGP-mediated downregulation of CCR4 along with its ligand CCL22 restricts Treg migration at the tumor site. NLGP is not apoptotic to Tregs but significantly downregulates the expression of Foxp3, CTLA4 and GITR. It also reverses the functional impairment of T-effector cells by Tregs, in terms of IFN-γ secretion, cellular proliferation and tumor cell cytotoxicity. NLGP also facilitates reconditioning of tumor microenvironment (hostile) by increasing IFN-γ and IL-12 but decreasing IL-10, TGF-ß, VEGF and IDO, creating an antitumor niche. Interaction between Foxp3, p-NFATc3 and p-Smad2/3, needed for successful Treg function, is also inhibited by NLGP. CONCLUSION: All of these coordinated events might result in inhibition of Treg associated-tumor growth and therefore increased survivability of mice having NLGP treatment before or/and after tumor inoculation. Thus, the possibility of NLGP being an excellent tool as a T-cell anergy breaker by abrogating the suppressor functions of Tregs in cancer needs to be explored further in the clinic.

Neem leaf glycoprotein matures myeloid derived dendritic cells and optimizes anti-tumor T cell functions.

In an objective to find out an effective, nontoxic dendritic cell (DC) maturating agent for human use, CD14(+) monocytes were differentiated with GMCSF/IL-4 and matured with neem leaf glycoprotein (NLGP). NLGP matured DCs (NLGP-DCs) show upregulated expression of CD83, CD80, CD86, CD40 and MHCs, in a comparable extent of control, LPS. NLGP-DCs secrete high amount of IL-12p70 with low IL-10. NLGP upregulates the expression of crucial transcription factor, ikaros, indicating maturation towards DC1 phenotype. Increased expression of CD28 and CD40L on T cells following co-culture with NLGP-DCs was noticed to promote DC-T interactions. As a result, T cells secrete high amount of IFN gamma with low IL-4 and generates anti-tumor type 1 immune microenvironment. Such NLGP-DCs present carcinoembryonic antigen (CEA) effectively to T cells to increase T cell mediated cytotoxicity of CEA(+) tumor cells in vitro and in vivo. With emergence of the NLGP as a promising DC maturating agent, NLGP-DCs can be used as a candidate vaccine tool for antigen specific cancer immunotherapy.

Restoration of dysregulated CC chemokine signaling for monocyte/macrophage chemotaxis in head and neck squamous cell carcinoma patients by neem leaf glycoprotein maximizes tumor cell cytotoxicity.

Previous studies have shown that the CC chemokine receptor CCR5 is downregulated on monocyte/macrophage (MO/Mphi) surfaces in head and neck squamous cell carcinoma (HNSCC) patients (stage IIIB). Ligands (RANTES, MIP-1alpha and MIP-1beta) of this chemokine receptor were also secreted in lesser quantity from MO/Mphi of HNSCC patients in comparison with healthy individuals. In an aim to restore this dysregulated receptor-ligand signaling, we have used neem leaf glycoprotein (NLGP), a novel immunomodulator reported from our laboratory. NLGP upregulated CCR5 expression, as evidenced from studies on MO/Mphi of peripheral blood from HNSCC patients as well as healthy individuals. Expression of RANTES, MIP-1alpha and MIP-1beta was also upregulated following NLGP treatment of these cells in vitro. Interestingly, NLGP has little effect on the expression of CCR5 and the ligand RANTES in oral cancer cells. This restored CCR5 receptor-ligand signaling seen in MO/Mphi was reflected in improved CCR5-dependent, p38 mitogen-activated protein kinase (MAPK)-mediated migration of MO/Mphi after NLGP treatment to a standard chemoattractant. NLGP also induces better antigen presentation and simultaneous costimulation to effector T cells by MO/Mphi by upregulating human leucocyte antigen (HLA)-ABC, CD80 and CD86. In addition, NLGP-treated MO/Mphi-primed T cells can effectively lyse tumor cells in vitro. The effects of NLGP on monocyte migration and T cell-mediated oral tumor cell killing were further demonstrated in transwell assays with or without CCR5 neutralization. These results suggest a new approach in cancer immunotherapy by modulating dysregulated CCR5 signals from MO/Mphi.

Induction of type 1 cytokines during neem leaf glycoprotein assisted carcinoembryonic antigen vaccination is associated with nitric oxide production.

Involvement of the nitric oxide (NO) release in CEAM phi NLGP (carcinoembryonic antigen pulsed macrophages with neem leaf glycoprotein) vaccination and its relationship with vaccine induced type 1 immune response were aimed to study in the present communication. Vaccination with CEAM phi NLGP resulted in macrophage activation as evidenced by its increased number and expression of CD69 marker. Activated macrophages demonstrated upregulation in synthesis of IL-12 and downregulation in IL-10, along with excess IFN gamma production in splenic cells, as evidenced from mRNA analysis. Induction of such type 1 immunity was further confirmed by expression of type 1 specific transcription factor, T-bet and enhancement of intracellular glutathione content. Such vaccination also induced greater nitric oxide (NO) production from macrophages. Dependence of induced type 1 immune response on the NO release and vice versa was studied by in vitro neutralization of IFN gamma/IL-12 and in vivo inhibition of NO production by methylene blue. Obtained results clearly demonstrated the interdependence of two anti-tumor immune functions, namely, NO production and generation of type 1 immune response. Understanding of the mechanism of this NO related immune modulation would have great impact in proposing CEAM phi NLGP vaccine in clinic for the treatment of CEA+ tumors.

Neem leaf glycoprotein induces perforin-mediated tumor cell killing by T and NK cells through differential regulation of IFNgamma signaling.

We have demonstrated augmentation of the CD3-CD56+ natural killer (NK) and CD8+CD56_ T-cell-mediated tumor cell cytotoxicity by neem leaf glycoprotein (NLGP). These NK and T cells were isolated from the peripheral blood of head and neck squamous cell carcinoma patients with a state of immunosuppression. NLGP induces TCRalphabeta-associated cytotoxic T lymphocyte (CTL) reaction to kill oral cancer (KB) cells. This CTL reaction is assisted by NLGP-mediated up-regulation of CD28 on T cells and HLA-ABC, CD80/86 on monocytes. CTL-mediated killing of KB cells and NK-cell-mediated killing of K562 (erythroleukemic) cells are associated with activation of these cells by NLGP. This activation is evidenced by increased expression of early activation marker CD69 with altered expression of CD45RO/CD45RA. NLGP is a strong inducer of IFNgamma from both T and NK cells; however, IFNgamma regulates the T-cell-mediated cytotoxicity only without affecting NK-cell-mediated one. Reason of this differential regulation may lie within up-regulated expression of IFNgamma-receptor on T-cell surface, not on NK cells. This NLGP-induced cytotoxicity is dependent on up-regulated perforin/granzyme B expression in killer cells, which is again IFNgamma dependent in T cells and independent in NK cells. Although, FasL expression is increased by NLGP, it may not be truly linked with the cytotoxic functions, as brefeldin A could not block such NLGP-mediated cytotoxicity, like, concanamycin A, a perforin inhibitor. On the basis of these results, we conclude that NLGP might be effective to recover the suppressed cytotoxic functions of NK and T cells from head and neck squamous cell carcinoma patients.

Neem leaf glycoprotein helps to generate carcinoembryonic antigen specific anti-tumor immune responses utilizing macrophage-mediated antigen presentation.

In an objective to generate effective carcinoembryonic antigen (CEA) specific anti-tumor immune response in Swiss mice, CEA was presented using macrophages with adjuvant help from neem leaf glycoprotein (NLGP). Such vaccination generates significantly higher antibody (IgG2a) and T cell response than immunization protocol without NLGP. NLGP controls the function of both B cells and macrophages by altering the expressions of various regulatory molecules, like, CD19, CD11b, etc. NLGP also directs CEA vaccination towards Th1 bias, by modulating cytokine secretion. This NLGP-generated anti-CEA immune response would be effective as a vaccine to lyse CEA(+) tumors in vitro and in vivo.