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.

Therapeutic anti-NLGP monoclonal antibody for carcinoembryonic antigen expressing tumors is nontoxic to Swiss and BALB/c mice.

A murine monoclonal antibody (mAb), 1C8 was developed against a novel glycoprotein NLGP and its unique property to recognize carcinoembryonic antigen (CEA) was reported. Utilizing this CEA recognizing property, 1C8 is successful to restrict the growth of CEA(+) murine and human cancers both in vitro and in vivo. Here, we have thoroughly evaluated the toxicity profile of this mAb 1C8 on different physiological systems of both tumor-free and tumor-bearing Swiss and BALB/c mice. Effective concentration (25 µg/mice) of 1C8 caused no behavioral changes in animals and no death was recorded. Moreover, little increase in the body and organ weights in all mice groups was noted. MAb 1C8 showed no adverse effect on the hematological system, but little hematostimulation was noticed, as evidenced by increased hemoglobin content, leukocyte count and lymphocyte numbers. Liver enzymes like alkaline phosphatase, SGOT, SGPT and nephrological products like urea and creatinine assessment confirmed no abnormalities in both hepatic and renal functions. Number of T cells, B cells, NK cells, macrophages and dendritic cells was upregulated in vivo by mAb treatment with significant downregulation of regulatory T cells. During this treatment serum levels of type 1 cytokines were upregulated over type 2 cytokines. This mAb 1C8 also did not induce any significant increase in antibody titer following treatment. Accumulated evidences from Swiss and BALB/c mice strongly suggest that this mAb 1C8 is completely safe, thus, can be recommended for further clinical trial for the therapy of CEA(+) tumors.

A monoclonal antibody against neem leaf glycoprotein recognizes carcinoembryonic antigen (CEA) and restricts CEA expressing tumor growth.

Carcinoembryonic antigen (CEA) is one of the promising tumor antigens mainly associated with carcinoma of the colon, lung, breast, etc. and received wide attention for cancer immunotherapy. Neem leaf glycoprotein (NLGP), an effective immunomodulator, is able to generate humoral and cellular immune responses in murine tumor models. We have generated a monoclonal antibody (mAb) against NLGP by fusing NLGP-immunized mice splenocytes with nonsecretory myeloma cells. A highly anti-NLGP mAb secreting clone (1C8; IgG2a in nature) has been identified and propagated in culture. 1C8 recognizes human CEA as good as NLGP by enzyme linked immunosorbent assay, Western blotting, and immunoprecipitation. 1C8 detects CEA on colon cancer tissues by immunochistochemistry. By flow cytometry, 1C8 specifically reacts with CEA(+) human (Colo-205, HCT-116, and HT-29) and mouse (CT-26) colon cancer cells, but it showed minimum reactivity with CEA(-) human (MCF7, SiHa, and SCC084) and mouse (B16MelF10) cancer cells. This anti-NLGP 1C8 mAb revealed significant antitumor activity and better survivability in vivo in animals bearing mouse (CT-26 in BALB/c) and human (Colo-205 in athymic nude) CEA(+) cancer cells. 1C8 has no direct influence on proliferation and migration of CEA(+) cells, however, NK cell-dependent strong antibody-dependent cellular cytotoxicity reaction toward CEA(+) cells and normalization of angiogenesis are chiefly associated with tumor growth restriction. Obtained results provided a new immunotherapeutic approach for the effective management of CEA(+) tumors.

Murine carcinoma expressing carcinoembryonic antigen-like protein is restricted by antibody against neem leaf glycoprotein.

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.

Neem leaf glycoprotein is superior than cisplatin and sunitinib malate in restricting melanoma growth by normalization of tumor microenvironment.

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, TGFß, 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.

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 is nontoxic to physiological functions of Swiss mice and Sprague Dawley rats: histological, biochemical and immunological perspectives.

We have evaluated the toxicity profile of a unique immunomodulator, neem leaf glycoprotein (NLGP) on different physiological systems of Swiss mice and Sprague Dawley rats. NLGP injection, even in higher doses than effective concentration caused no behavioral changes in animals and no death. NLGP injection increased the body weights of mice slightly without any change in organ weights. NLGP showed no adverse effect on the hematological system. Moreover, little hematostimulation was noticed, as evidenced by increased hemoglobin content, leukocyte count and lymphocyte numbers. Histological assessment of different organs revealed no alterations in the organ microstructure of the NLGP treated mice and rats. Histological normalcy of liver and kidney was further confirmed by the assessment of liver enzymes like alkaline phosphatase, SGOT, SGPT and nephrological products like urea and creatinine. NLGP has no apoptotic effect on immune cells but induces proliferation of mononuclear cells collected from mice and rats. Number of CD4(+), CD8(+) T cells, DX5(+) NK cells, CD11b(+) macrophages and CD11c(+) dendritic cells is upregulated by NLGP without a significant change in CD4(+)CD25(+)Foxp3(+) regulatory T cells. Type 1 cytokines, like IFNγ also increased in serum with a decrease in type 2 cytokines. Total IgG content, especially IgG2a increased in NLGP treated mice. These type 1 directed changes help to create an anti-tumor immune environment that results in the restriction of carcinoma growth in mice. Accumulated evidence strongly suggests the non-toxic nature of NLGP. Thus, it can be recommended for human use in anti-cancer therapy.

IFNα2b augments immune responses of cisplatin+5-fluorouracil treated tongue squamous cell carcinoma patients--a preliminary study.

BACKGROUND & OBJECTIVES: Interferon alpha 2b (IFNα2b) has been reported to regulate several immune functions efficiently to enhance the cytotoxic activity of NK and T cells towards various forms of tumours. The objective of the present study was to evaluate the efficacy of IFNα2b in overcoming disease induced and/or treatment associated imunosuppression of tongue squamous cell carcinoma (TSCC) patients undergoing chemotherapy for better clinical outcome. METHODS: Seven TSCC patients under cisplatin + 5-fluorouracil chemotherapy in combination with IFNα2b were assessed for various immunohaematological parameters before treatment, after chemotherapy and after IFNα2b therapy. RESULTS: Deterioration of the haematological and immune responses was detected in immunosuppressed TSCC patients after chemotherapy. IFNα2b treatment led to a recovery in these parameters in most of the patients. Greater number of T/NK cells and enhanced secretion of type 1 cytokines were also noted. Haematological complications were reduced after completion of the therapy. Immune- and haematostimulation were also observed in patients with partial response. No positive clinical response was detected in one patient. INTERPRETATION & CONCLUSIONS: IFNα2b appears to be an effective immunostimulator having clinical impact to combat the immunosuppression in TSCC patients. Successful immunostimulation by IFNα2b may help TSCC patients in clinical improvement. The findings of this preliminary study need to be confirmed on a large number of patients with TSCC.

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 partially rectifies suppressed dendritic cell functions and associated T cell efficacy in patients with stage IIIB cervical cancer.

Myeloid-derived dendritic cells (DCs) generated from monocytes obtained from stage IIIB cervical cancer (CaCx IIIB) patients show dysfunctional maturation; thus, antitumor T cell functions are dysregulated. In an objective to optimize these dysregulated immune functions, the present study is focused on the ability of neem leaf glycoprotein (NLGP), a nontoxic preparation of the neem leaf, to induce optimum maturation of dendritic cells from CaCx IIIB patients. In vitro NLGP treatment of immature DCs (iDCs) obtained from CaCx IIIB patients results in upregulated expression of various cell surface markers (CD40, CD83, CD80, CD86, and HLA-ABC), which indicates DC maturation. Consequently, NLGP-matured DCs displayed balanced cytokine secretions, with type 1 bias and noteworthy functional properties. These DCs displayed substantial T cell allostimulatory capacity and promoted the generation of cytotoxic T lymphocytes (CTLs). Although NLGP-matured DCs derived from CaCx monocytes are generally subdued compared to those with a healthy monocyte origin, considerable revival of the suppressed DC-based immune functions is noted in vitro at a fairly advanced stage of CaCx, and thus, further exploration of ex vivo and in vivo DC-based vaccines is proposed. Moreover, the DC maturating efficacy of NLGP might be much more effective in the earlier stages of CaCx, where the extent of immune dysregulation is less and, thus, the scope of further investigation may be explored.

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.

Association of early phase of colorectal carcinogenesis with STAT3 activation and its relevance in apoptosis regulation.

Expression of STAT3/pSTAT3 in colorectal cancer (CRC) patients of Indian origin was studied to assess its significance in early detection and apoptosis regulation. Colorectal tissues with malignant lesions were STAT3/pSTAT3 positive in 66% of the cases and among these positive cases, well differentiated, moderately differentiated and poorly differentiated cancers were 86%, 60% and 0% respectively. All CRC specimens studied were immunoreactive with anti-carcinoembryonic antigen antibody. Cells purified from CRC tissues exhibited greater STAT3/pSTAT3 reactivity than peripheral blood mononuclear cells (PBMC) from healthy individuals, which served as control. apoptotic index (AI) was comparatively low in tissue specimens with STAT3/pSTAT3 expression. CRC cells with a comparatively less number of apoptotic cells, expressed a minimum number of Caspase-3 positive cells (4.73%), in comparison to healthy-PBMC (12.63%). CRC cells with high STAT3/pSTAT3 staining had cells with greater percentage of Bcl2 reactivity (23.05%), but less positivity with Caspase3 antibody (2.05%). Overall data suggests that CRC population was STAT3/pSTAT3 immunoreactive in a stage specific manner and STAT3 protects cancerous colorectal epithelial cells from apoptosis. Bcl-2, Cyclin D1 and Caspase-3 control the activity of apoptosis regulator, STAT3.

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.

The role of copper in drug-resistant murine and human tumors.

Multidrug resistance (MDR) is still a major threat to successful clinical application of cancer chemotherapy. Copper plays an important role in biological systems, and copper is also involved in carcinogenesis. In the present investigation, we addressed the question whether metal copper might be involved in drug resistance of murine and human tumors. By means of atomic absorption spectroscopy, we determined serum copper concentrations. We found that the blood serum of tumor-bearing mice contained higher amounts of copper than healthy mice with tumors. Secondly, mice bearing doxorubicin-resistant Ehrlich ascites carcinoma- or cyclophosphamide-resistant Lewis lung carcinoma contained more copper in their serum than mice bearing the corresponding drug-sensitive parental tumors. Furthermore, the analysis of patients with breast cancer, colon carcinoma or lung cancer showed that the serum copper contents were higher in patients not responding to chemotherapy when compared to patients whose tumors responded to treatment. The copper levels in serum of healthy volunteers were lower than in cancer patients irrespective of their response to chemotherapy. Our results imply that the level of serum copper may be considered as a biomarker for treatment response.

Interferon-alpha2b restores the impaired chemotactic activity of peripheral blood mononuclear cells from head and neck squamous cell carcinoma patients by modulating CXC receptor ligand interaction.

We have studied the immunomodulatory effects of interferon-alpha2b (IFN-alpha2b) in rectification of the dysregulated IFN-gamma-dependent chemokines and their receptor CXCR3 splice variants in head and neck squamous cell carcinoma-peripheral blood mononuclear cells (HNSCC-PBMC). CXCR3 expression was upregulated in HNSCC-PBMC, with the demonstration of poor chemotactic function. CXCR3 upregulation possibly represents the increased synthesis of CXCR3B splice variant, without significant change in CXCR3A. Upregulated expression of CXCR3B was downregulated following in vitro IFN-alpha2b treatment of HNSCC-PBMC. Upregulation of CXCR3A+B by IFN-alpha2b with downregulation of the CXCR3B indirectly suggests that the upregulation of the CXCR3A splice variant induces cellular migration. On the other hand, the stimulation of PBMC with IFN-alpha2b maintains physiological homeostasis of CXCR3 ligands, CXCL10 and CXCL9, and increases the secretion of IFN-gamma. The suppressed chemotactic ability of HNSCC-PBMC could be restored either by in vitro treatment of PBMC with IFN-alpha2b or during the use of IFN-alpha2b stimulated PBMC supernatant as a chemoattractant. Chemoattraction process is guided at the level of both receptor and its ligands, as confirmed by neutralization studies. IFN-alpha2b possibly controls chemotaxis by regulating the interaction between CXCL10 and CXCR3A. Neutralization of IFN-gamma downregulates the IFN-alpha2b mediated CXCL10 release, suggesting the active role of IFN-gamma in the transduction of chemotactic signal for the migration of cytotoxic T/NK cells at the tumor site.

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 restores the impaired chemotactic activity of peripheral blood mononuclear cells from head and neck squamous cell carcinoma patients by maintaining CXCR3/CXCL10 balance.

Interaction between CXCL10 and CXCR3 is dysregulated in head and neck squamous cell carcinoma (HNSCC) and hampers chemotaxis of cytotoxic cells at tumor site. In continuation of the demonstration of significant immunomodulatory effects of neem leaf preparation (NLP), the active ingredient of NLP is characterized as a glycoprotein (NLGP). NLGP is responsible for in vivo immunomodulation to restrict the growth of mice tumors. Effect of NLGP in rectification of the dysregulated IFN gamma dependent chemokine and its receptor CXCR3 splice variants was investigated. Upregulated expression of CXCR3B in HNSCC-PBMC were downregulated following in vitro NLGP treatment. Unchanged expression of CXCR3A+B by NLGP with downregulation of the CXCR3B indirectly suggests the upregulation of the CXCR3A, responsible for cellular migration. However, stimulation of healthy-PBMC with NLGP maintains physiological homeostasis of CXCL10 and increases IFN gamma secretion. The suppressed chemotaxis of HNSCC-PBMC could be restored either by in vitro treatment with NLGP or during use of NLGP stimulated PBMC supernatant as a chemoattractant. Neutralization studies confirmed that the chemoattraction process is guided by both receptor (CXCR3A) and its ligand (CXCL10). Neutralization of the IFN gamma in PBMC culture in presence of NLGP unexpectedly increases the intracellular release of CXCL10, suggesting the NLGP mediated IFN gamma independent release of CXCL10. Interestingly, downregulation of the CXCL10 release was detected after IFN gamma neutralization in absence of NLGP and IFN gamma receptor neutralization in presence of NLGP. Efficacy of NLGP in restoration of the dysregulation of the chemokine signaling may be utilized to design new immunotherapeutic protocol.

Generation of Her-2/neu vaccine utilizing idiotypic network cascade.

Our goal is to apply an anti-idiotype (Id) antibody based vaccine approach for the treatment of Her-2/neu-positive human cancer. Amplification and/or overexpression of Her-2/neu occur in multiple human malignancies and are associated with poor prognosis. The Her-2/neu proto-oncogene is a suitable target for cancer immunotherapy. Our strategy is active specific immunotherapy in which patients immunized with an anti- Id antibody mimicking Her-2/neu will generate sustained high titer Her-2/neu specific protective antibodies. We have used an anti-Her-2/neu murine monoclonal antibody 4D5 as the immunizing antibody (Ab1) against which monoclonal anti-Ids or Ab2s were generated in syngeneic mice. We have characterized one such anti-Id (Ab2) designated 6D12, which mimics a specific epitope of Her-2/neu as defined by Trastuzumab, and can be used as a surrogate antigen for Her-2/neu across the species barriers. Immunization of allogeneic mice or rabbits with 6D12 induced anti-anti-Id (Ab3), that specifically recognized Her-2/neu-positive tumor cells and lysed these cells in culture by antibody-dependent cellular cytotoxicity (ADCC). Monoclonal Ab3 generated in mice against 6D12 inhibited the proliferation of Her-2/neu-positive SK-BR-3 cells in vitro in a dose dependent fashion and delayed the growth of Her-2/neu transfected EL4-Her-2 cells in vivo. These data suggest the potential use of 6D12 as a vaccine for Her-2/neu-positive human cancer.

Anti-tumor immunity induced by an anti-idiotype antibody mimicking human Her-2/neu.

Our goal is to apply an anti-idiotype (Id) antibody based vaccine approach for the treatment of Her-2/neu-positive human cancer. Amplification and/or over-expression of Her-2/neu occur in multiple human malignancies and are associated with poor prognosis. Her-2/neu proto-oncogene is a suitable target for cancer immunotherapy. We have developed and characterized a murine monoclonal anti-Id antibody, 6D12 that mimics a specific epitope of Her-2/neu and can be used as a surrogate antigen for Her-2/neu. In this study, the efficacy of 6D12 as a tumor vaccine was evaluated in a murine tumor model. Immunization of immunocompetent C57BL/6 mice with 6D12 conjugated to keyhole limpet hemocyanin and mixed with Freund's adjuvant or 6D12 combined with the adjuvant QS21 induced anti-6D12 as well as anti-Her-2/neu immunity. Her-2/neu-positive human breast carcinoma cells, SK-BR-3 reacted with immunized mice sera as determined by ELISA and flow cytometry. Flow cytometry analysis also demonstrated strong reactivity of immunized mice sera with human Her-2/neu transfected EL4 cells (EL4-Her-2), but no reactivity with nontransfected parental EL4 cells. Antibody dependent cellular cytotoxicity against EL4-Her-2 cells was also observed in presence of immune sera. Mice immunized with 6D12 were protected against a challenge with lethal doses of EL4-Her-2 cells, whereas no protection was observed against parental EL4 cells or when mice were immunized with an unrelated anti-Id antibody and challenged with EL4-Her-2 cells. These data suggest that anti-Id 6D12 vaccine can induce protective Her-2/neu specific antitumor immunity and may serve as a potential network antigen for the treatment of patients with Her-2/neu-positive tumors.