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

Vaccine 28 (2010) 1241–1252

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Neem leaf glycoprotein matures myeloid derived dendritic cells and optimizes anti-tumor T cell functions Shyamal Goswami a , Anamika Bose a , Koustav Sarkar a , Soumyabrata Roy a , Tathagata Chakraborty a , Utpal Sanyal b , Rathindranath Baral a,∗ a b

Department of Immunoregulation and Immunodiagnostics, Chittaranjan National Cancer Institute (CNCI), 37, S. P. Mukherjee Road, Kolkata, West Bengal 700026, India Anti Cancer Drug Development, Chittaranjan National Cancer Institute, 37, S. P. Mukherjee Road, Kolkata 700026, India

a r t i c l e

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Article history: Received 9 July 2009 Received in revised form 21 October 2009 Accepted 8 November 2009 Available online 5 December 2009 Keywords: Carcinoembryonic antigen Dendritic cells Neem leaf glycoprotein

a b s t r a c t 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␥ 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. © 2009 Elsevier Ltd. All rights reserved.

1. Introduction Dendritic cells (DCs) are professional antigen presenting cells (APC), continuously generated from hematopoietic stem cells [1] to function in identifying microbial structure, tumor antigens, etc. and to initiate immune responses by priming naïve T cells [2]. DCs play pivotal role not only in the initiation but also in the determining direction (either type 1 or 2) of T cell mediated immune responses [3]. DCs are widely distributed as immature DCs (iDCs), into both lymphoid and nonlymphoid tissues [4,5]. Immature DCs can take up foreign antigens, but cannot present it into T cells to induce effective T cell immunity [6]. In response to pathogens, DCs undergo differentiation from iDC to mature immunogenic DCs (mDCs) [7]. These mDCs express high amount of cell surface major histocompatibility complexes (MHCs) and co-stimulatory molecules [8,9], accordingly efficiently prime primary T cell mediated immune reactions. Recent strategies for developing preventive and therapeutic vaccines have focused on the ability of mDCs to deliver antigens as antigenic peptides to CD4+ and CD8+ T lymphocytes [4,10,11]. The field of DC targeting has been dominated largely by ex vivo strategies, consist of isolating DCs from blood, exposing them

to maturating stimuli with or without antigen and re-injecting them into patients [12]. For this purpose, a nontoxic maturating agent is requisite. In general, lipopolysaccharide (LPS) is an agent used for ex vivo DC maturation [13]. In addition to LPS, type 1 interferons (IFN␣/␤) [14], TNF␣ [15], Flt3 ligand [16] and CpG oligodeoxynucleotide (CpGODN) [17] play the role of DC maturating agent. Toxicities are major limitations of some agents, like, LPS and TNF␣ [18]. Additionally, LPS is an inducer of immunosuppressive cytokine, IL-10 [19]. Flt3 ligand and CpGODN are comparatively nontoxic [20], but little cost effective to use in third world countries, like, India. In this context, candidature of neem leaf glycoprotein (NLGP) is examined as a DC maturating agent. This selection is based on our earlier observation, where rich source of NLGP, neem leaf preparation, was shown to be effective to release a good amount of IL-12 [21]. Data presented here indicate that GMCSF/IL-4 differentiated DCs, when matured with NLGP, are highly effective means for the generation of optimum type 1 T cell response showing anti-tumor functions in vitro and in vivo. 2. Materials and methods 2.1. Reagents and antibodies

∗ Corresponding author. Tel.: +91 033 2476 5101x334; fax: +91 033 2475 7606. E-mail addresses: [email protected], [email protected] (R. Baral). 0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.11.018

Recombinant human and mouse GMCSF and IL-4 (rhGMCSF, rmGMCSF, rhIL-4, rmIL-4) were obtained from BD-Pharmingen

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(San Diego, CA). Fluorescence labeled anti-human CD1a, CD3, CD4, CD8, CD14, CD40L, CD69, CD45RA, CD45RO, and CD62L monoclonal antibodies were procured from BD-Pharmingen (San Diego, CA). Fluorescence labeled anti-human CD28, CD40, CD80, CD86, CD83, HLA-ABC and anti-mouse CD80, CD86, MHC-II, CD11c were obtained from e-Biosciences (San Diego, CA). Anti-ikaros antibody was purchased from Santa-Cruz, California, USA. OptEIATM kits for cytokine estimation (IFN␥, IL-12, IL-10 and IL-4) were obtained from BD-Pharmingen (San Diego, CA). Kits for LDH release assay for cytotoxicity detection and chemiluminescence for immunoblotting were obtained from Roche Diagnostics, Mannheim, Germany. LPS and LPS inhibitor, polymixin B, were procured from Sigma, St. Louis, USA. Lymphocyte separation media was purchased from MP Biomedicals (Solan, OH). 2.2. Neem leaf glycoprotein (NLGP) Extract from neem (Azadirachta indica) leaves was prepared by the method as described earlier [22,23]. Mature leaves of same size and color (indicative of same age), taken from a standard source were shed-dried and pulverized. Leaf powder was soaked overnight in phosphate buffered saline (PBS); pH 7.4, supernatant was collected by centrifugation at 1500 rpm. Neem leaf preparation (NLP) was then extensively dialyzed against PBS, pH 7.4 and concentrated by Centricon Membrane Filter (Millipore Corporation, Bedford, MA, USA) with 10 kDa molecular weight cut off. Endotoxin content of the freshly prepared NLP was determined by Limulus Amebocyte Lysate (LAL) test as per manufacturer’s (Salesworth India, Bangalore) instruction. The endotoxin content of all the batches of NLP (extract obtained from 0.25 mg leaf powder in 1 ml PBS) was found to be less than 6 pg/ml. Glycoprotein present in this NLP (Neem leaf glycoprotein-NLGP) was isolated and characterized by the method described [24,25]. The purity of NLGP was checked by Size exclusion-HPLC (SEHPLC) in a protein PAK 300 SW column of 7.5 mm (ID) × 30 cm. The glycoprotein was eluted using gradients of PBS at a constant flow rate of 1.0 ml/min under a pressure of 3 × 106 N/m2 . We used 20 ␮g of BSA (Sigma) to standardize the retention time of the protein within the column. The protein peaks were determined by absorption at 280 nm in a UV recorder. 2.3. Animals and tumor cells Female Swiss mice (age, 6–8 weeks, body weight, 24–27 g) were obtained from Institutional Animal Facilities, CNCI, Kolkata, India. Autoclaved dry pellet diet (Epic Laboratory Animal Feed, Kalyani, India) and water were given ad libitum. Maintenance and treatment of animals were given according to the guidelines established by the Institutional Animal Care and Ethics Committee. Colorectal tumors were developed in Swiss mice, using 20-methylcholanthrene according to the method described [24,26]. Tumors were removed from tumor bearing mice, minced to prepare the tumor cell suspension and 1 × 106 cells were injected subcutaneously into syngenic mice and allowed to grow as solid tumor. Expression of CEA on these mouse tumor cells was determined using anti-CEA antibody by flow cytometry.

2.5. Generation and maturation of DCs Human peripheral blood was collected from male healthy volunteers of the age group 25–30 after obtaining their informed consent. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation over lymphocyte separation media. Myeloid DCs were generated from the CD14+ adherent fraction of PBMC (plastic adherence of monocytes, cellular purity was checked by flow cytometry) by culturing in six well plates (1.0 to 1.5 × 106 cells/ml) with complete RPMI1640 medium with 10% FBS, supplemented with rhGMCSF (1000 U/ml) and rhIL-4 (500 U/ml) for 5 days, then cultured for additional 3 days with either LPS (1 ␮g/ml) or NLGP (1.5 ␮g/ml). Every 2 days the media including the supplements were refreshed. After 8 days of culture, mDCs were harvested and extensively washed before use. Following 6 days or 8 days of DC culture, supernatants (stored at −80 ◦ C) were used for the estimation of IL-12p70 and IL-10. Primary mouse bone marrow DCs were obtained from Swiss mice according to the protocol described [27]. In brief, single-cell suspension was obtained after flushing bone marrow from the tibia and femurs. Erythrocytes were lysed by resuspending the cell pellet in a hypotonic buffer. The cells were cultured (2 × 106 cells/well) with complete RPMI 1640 medium containing 10% FBS, 10 ng/ml rmGMCSF and 5 ng/ml rmIL-4. On day 6 of culture, nonadherent cells obtained from these cultures were considered to be immature BmDC. For maturation, LPS and NLGP were added separately for the final 2 days of cell culture. 2.6. MACS purification of CD8+ T cells CD8+ T cells were purified using magnetic activated cell sorter (MACS) (Milltani Biotech Inc., CA, USA). In brief, nonadherent mononuclear cells were labeled with cocktail of antibodies (except CD8+ antibodies), conjugated with magnetic beads and passed through the magnetic column. Flow through enriched with CD8+ cells were collected as pure CD8+ T cell population. Cells were washed and used in various functional assays of T cells. The isolated T cell populations exceeded 95% purity as assessed by flow cytometry (data not shown). 2.7. Flow cytometric analysis of immune cellular markers Flow cytometric analysis for surface phenotypic markers for monocytes, iDCs and mDCs were performed after labeling with 20 ␮l of different anti-human fluorescence labeled antibodies (CD1a, CD3, CD8, CD28, CD14, CD45RA, CD45RO, CD69, CD62L, CD80, CD86, CD83 and HLA-ABC) for 30 min as per manufacturer’s recommendation. After labeling, cells were washed in FACS buffer (PBS with 1% FBS), fixed in 1% paraformaldehyde in PBS and cytometry was performed by using Cell Quest software on a FACScan flow cytometer (Becton Dickinson, Mountainview, CA). Suitable negative isotype controls were used to rule out the background fluorescence. Data was analyzed by FlowJo software in some cases. The data was generated by cytofluorometric analyses of 10,000 events. Percentage of each positive population and mean fluorescence intensity (MFI) were determined by using quadrant statistics. Similarly, mouse BmDCs were stained with anti-mouse fluorescence labeled CD11c, CD80, CD86, and MHC-II and analysed by flow cytometer.

2.4. Tumor cell lines The CEA+ human colon cancer (COLO205) and CEA− human oral cancer (KB) cell lines originally obtained from the National Center for Cell Sciences, Pune, were maintained in RPMI 1640 (Life Technologies, NY) with 10% FBS, penicillin (50 units/ml) and streptomycin (50 ␮g/ml) at 37 ◦ C with the supply of 5% CO2 .

2.8. Mixed lymphocyte reaction (MLR) with CD8+ T cells for proliferation and cytokine secretion Purified CD8+ T cells were used for allogeneic MLR assays. For proliferation and cytokine secretion, irradiated (3000 rad) mDCs were co-cultured with allogenic CD8+ T cells at DC:T cell ratios

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of 1:10, 1:50 and 1:100. Supernatants were collected for cytokine assays after 72 h and proliferation was checked after 96 h by MTT assays as described earlier [22]. 2.9. Mixed lymphocyte reaction (MLR) with CD8+ T cells for CEA specific cytotoxicity For cytotoxicity assay, mDCs matured with either LPS or NLGP, were pulsed with CEA and irradiated (3000 rad) to co-culture with autologous and allogenic T cells. Cytotoxic T lymphocytes (CTLs) generated from these cultures (1 × 105 ) were incubated with CEA+ COLO205 and CEA− KB cells (1 × 104 ) for overnight in different E:T ratio (1:10, 1:50, 1:100) to obtain CTL mediated cytotoxicity of tumor cells. The cytotoxicity was tested by LDH release assay [24] using cytotoxicity detection kit. 2.10. Cytokine detection assays To quantify cytokines, supernatants from mDC cultures and MLRs were harvested at different time points and filtered (0.2 ␮m filters). Secretion of IFN␥, IL-12p70, IL-10 and IL-4 was assessed with ELISA kits as per manufacturer’s instruction and optical density was measured at 450 nm using microplate reader (BioTek Instruments Inc., Vermont, USA). 2.11. Immunocytochemistry for Ikaros expression DCs at its different phases of generation and maturation were studied for the expression of ikaros by immunocytochemistry. In brief, DCs were cytospined (SIGMA-3K10, Germany) over poly-llysine (Sigma, USA) coated slides, fixed and permeabilized in chilled methanol followed by acetone. After blocking with 5% BSA, the cytospined cells were incubated with anti-human rabbit ikaros primary antibody. Specific antibody binding was detected using anti-rabbit IgG-HRP (Sigma, St. Louis, USA). HRP reaction was detected by aminoethylcarbazol (AEC kit, Vector Laboratories Inc, USA). Slides were viewed under microscope (Leica DM-4000, Germany) after counterstaining with hematoxylin and photographed. Five representative fields were counted for the number of positive cells. Photographs were analyzed and staining intensity quantitated using Adobe Photoshop CS2 following the method outlined at http://www.lukemiller.org/journal/2007/08/quantifyingwestern blots-without.html. 2.12. Tumor growth restriction assay Four groups of Swiss mice (n = 6 in each group) were immunized with iDCs, LPS matured BmDCs, NLGP matured BmDCs and NLGP matured CEA pulsed BmDCs (2 × 105 cells in each case) weekly for three times in total. Three days following completion of the immunization, mice were inoculated with CEA+ tumor cells (1 × 107 ) subcutaneously on right hind leg quarter. Growth of solid tumor (in mm3 ) was monitored weekly by caliper measurement using the formula: (width2 × length)/2. Survival of mice was monitored regularly, till tumor size reached to 25 mm in either direction. Spleens from all four groups of mice on day 20, following tumor inoculation of an identical set of experiment were isolated, single cells prepared and incubated: (i) with irradiated tumor cells for 5 days, then, CD8+ T cells were purified by MACS column and again co-cultured with NLGP-DCs in presence of CEA and further incubated for 2 days. Supernatants from this culture were collected and cell free supernatants were assessed for IFN␥ release and (ii) for 72 h in presence of either CEA or ConA and finally cell proliferation was checked by MTT assay.

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2.13. Statistical analysis All results represent the average of 4–6 individual experiments involving triplicate assays. Statistical significance was established by unpaired t-test using INSTAT 3 Software. 3. Results 3.1. NLGP helps in DC maturation Purity of the NLGP used in this study was checked by SE-HPLC. An aliquot of NLGP (injection volume-20 ␮l) containing 1.4 ␮g of protein was subjected to pass through a protein PAK 300 SW column. The glycoprotein was eluted using 100 mM PBS obtaining a single peak having specific point RT at 11.945 min (Fig. 1A). As optimum maturation of DCs is crucial for their requisite functions [28], evaluation of the DC maturating ability of NLGP in comparison to LPS was performed on human peripheral blood monocyte (CD14+ ) culture in presence of GMCSF and IL-4 as differentiating agent (Fig. 1A.1). Following differentiation myeloid cells acquired the high expression of CD1a with downregulation of CD14 expression. GMCSF and IL-4 differentiated CD1ahigh immature DCs (iDC) expressed HLA-ABC, CD80 and CD86 (Fig. 2A and B). However, maturation with NLGP resulted with CD1alow DC with comparatively higher expression of MHCs, CD80 and CD86 (Fig. 2A and B). In addition, maturation caused acquisition of the expression of mDC specific maturation marker CD83 (Fig. 2B). Maturation of mouse BmDCs was monitored by studying the expression of CD11c and MHC-II on their cell surfaces and significant upregulation in these markers was noticed in NLGP matured BmDCs (Fig. 2C). BmDCs also showed higher expression of costimulatory molecules, CD80 and CD86 upon maturation with NLGP (Fig. 2C). Next, we have also studied the effect of NLGP on two different DC subsets, and NLGP have more prominent effect on CD8+ DCs, those are specialized for antigen presentation to CD8+ T cells through MHC class I molecules. NLGP treatment upregulates both number and MFI of CD8+ DCs. On the otherhand, NLGP increases only the percentage of CD8− DCs, which is specialized for antigen processing and presentation through MHC class-II molecules to CD4+ T cells, not the MFI (Fig. 2D). 3.2. NLGP polarizes dendritic cells towards type 1 phenotype Type 1 polarization of DCs is required for its optimum antitumor function, which is dependent on an exogenous IL-12 inducing factor [29]. To ascertain whether NLGP induces DC1 phenotype, release of IL-12 and IL-10 were assessed from cell free DC culture supernatant, following its maturation with NLGP and LPS (day 8). As presented in Fig. 3, increase in IL-12 with subsequent decrease in IL-10 was observed following maturation of DCs with NLGP. Supplementation of endotoxin inhibitor, polymixin B (50 ␮g/ml) in DC culture did not affect NLGP-induced IL-12 release from matured DCs (mDCs). On the otherhand, polymixinB downregulates LPS-induced IL-12 release from mDCs (Fig. 3). 3.3. NLGP-induced maturation is associated with ikaros expression Ikaros proteins are essential for cellular differentiation [30] and recently, it appeared as a crucial transcription factor for maturation towards DC1 phenotype. In order to find out their involvement in NLGP matured myeloid DCs, ikaros expression was studied by immunocytochemistry. Ikaros levels were below detection in human adherent blood monocytes (Fig. 4; day 0). However, differentiation of myeloid DC precursor cells into iDCs was associated

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Fig. 1. Purity of NLGP was analysed by SE-HPLC (A). Morphological changes and expression of CD1a, CD3 and CD14 in myeloid DCs during differentiation and maturation. (A.1) Adherent fraction (CD14+ ) of human PBMC was either kept untreated for 8 days, or cultured with GMCSF/IL-4 for 5 days, then, matured with either LPS or NLGP for 8 days in total. Microscopical views were presented as observed under 10× and 40× (inset) objectives. (B) Acquisition and loss of different markers during differentiation and maturation of CD14+ macrophages to DCs. Representative figures from flow cytometric analysis of staining for CD1a, CD14 and CD3 markers on monocytes, GMCSF/IL-4 differentiated DCs and LPS as well as NLGP matured DCs are presented. Oval, CD1alow cells; Rectangle, CD1ahigh cells.

with the induction of ikaros in the form of speckles in the nucleus in between day 6 and day 8 of culture. While, consistent with above mentioned results, in NLGP matured CD83high DCs, levels of ikaros subsequently increased with respect to number of positive cells and intensity (Fig. 4B). This increment is even greater than staining observed in LPS matured DC preparation (Fig. 4), establishing NLGP as a DC maturating agent, directing towards DC1 phenotype. 3.4. NLGP matured DCs induce optimum T cell functions Investigation on the role of mDCs, generated with NLGP, on T cell functions was carried out in MLRs. Co-culture of MACS purified CD8+ T cells with NLGP matured DCs caused significant lose of naïve T cell marker, CD45RA with subsequent gain of memory marker CD45RO on T cell surface confirming the capability of NLGP matured DCs to polarized resting naïve T cells towards effector or antigen specific memory phenotype. This culture condition also upregulated the expression of an early activation marker CD69 and downregulated lymphocyte homing receptor, CD62L (Fig. 5).

These evidences suggest T cell activation by mDCs and such T cell activation was more prominent when NLGP matured DCs were used in co-culture. NLGP generated mDCs induce optimum proliferation of purified CD8+ CD56− T cells and addition of NLGP in culture resulted further increase in T cell proliferation (Fig. 6A). Culture supernatants from T cell culture in presence of mDCs were assessed for cytokines, IFN␥ and IL-4. It is apparent from Fig. 6B that mDC assisted T cells released significantly higher amount of IFN␥ in presence or absence of NLGP. On the otherhand, similar culture condition released lower amount of IL-4 (Fig. 6B). 3.5. NLGP matured DCs help in T cell mediated cytotoxicity in vitro Flow cytometric analysis of CD28 on surface of T cells, cultured with mDCs matured with either NLGP or LPS, revealed higher expression of this participating molecule (Fig. 7A) in cytotoxic T lymphocyte (CTL) reaction. In this CTL reaction, participation of co-stimulatory molecules is essential and NLGP upregulates CD80, CD86 on the surface of mDCs (Fig. 2A and B).

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Fig. 2. Expression of co-stimulatory and maturation markers on human and mouse DC surfaces. Human myeloid cells (A and B) and bone marrow cells from Swiss mice (C and D) were differentiated with GMCSF/IL-4 and matured with either LPS or NLGP to DCs. Expression of CD1a/HLA-ABC, CD80/CD86 and CD83 in human system (A and B) and CD11c, MHC-II and CD80/CD86 in mouse system (C) were studied using various fluorescence labeled markers as indicated and analyzed by flow cytometry using Cell Quest and FlowJo software. An appropriate isotype-matched antibody was used as negative control (open histogram). The values indicated on the histograms are mean fluorescence intensities (MFI; upper right corner) of the positive cells (given as percent in M1) (B and C) in the gated population (A). Gating on figures showing HLA and CD1a expression designates CD1a high and low status (A). Dot plot on CD80/CD86 data from mice is presented (C, lower panel). The results shown were obtained from a single experiment with a single donor (A and B) and a mice (C and D) and are representative of 6 similar experiments (mean ± SD), as presented in bar diagram in each case. CD8 reactivity of iDC, mDC-LPS and mDC-NLGP was also demonstrated (D). *p < 0.001, in comparison to iDC.

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Fig. 2. (Continued ).

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Fig. 3. Type 1 polarization by NLGP matured DCs. (A) NLGP-induced type 1 cytokine secretion is independent of polymixin B sulfate. Human matured DCs were cultured for 7 days with LPS or NLGP. Polymixin B sulfate was administered in control wells along with LPS and NLGP. Secreted IL-12 and IL-10 were measured by ELISA in culture supernatants after 48 h. Data are the mean values of four independent experiments. *p < 0.0001.

Reflection of optimization of T cell functions by NLGP matured DCs on CTL mediated tumor cell killing was assessed in vitro. NLGP and LPS matured mDCs were pulsed with CEA and CEA pulsed irradiated mDCs were co-cultured with autologous and allogenic T cells in MLRs. Finally, cells of MLR were incubated with CEA+ COLO205 and CEA− KB cells. mDCs always promote the cytotoxicity of CEA+ tumor cells tested, in comparison to those cultures, where iDCs were supplemented (Fig. 7B). Again, these CTL reactions were more effective than those culture conditions using LPS matured DCs. Observed CTL reactions were downregulated significantly following blocking of TCR␣␤ and HLA-ABC (data not shown), indicating role of TCR activation in CEA presentation to T cells by NLGP matured DCs having high expression of CD80/CD86.

3.6. NLGP matured DCs help in the growth restriction of CEA+ mouse tumors In vitro participation of NLGP matured DCs in antigen specific CTL mediated killing prompted us to study such effects on in vivo system. Four groups of Swiss mice (n = 6 in each group) were immunized with either NLGP matured CEA pulsed BmDCs or NLGP matured BmDCs or LPS matured BmDCs or with iDCs in alternate weeks for three times in total. Three days following completion of the immunization, mice were inoculated with CEA+ colon tumor cells. Monitoring of tumor growth in all groups of mice clearly demonstrated that tumor growth was restricted in mice immunized with NLGP matured CEA pulsed BmDCs (Fig. 8A), in comparison to mice of other groups. NLGP matured BmDCs also

Fig. 4. Expression of ikaros protein is associated with NLGP-induced DC maturation. (A) Human myeloid cells were differentiated with GMCSF/IL-4 and matured with either LPS or NLGP to DCs. DCs at its state of differentiation and maturation was harvested and cytospined on glass slides. Adhered DCs were stained immunocytochemically using ikaros antibodies and colour was developed with aminoethylcarbazol. Upper panel, 10×; Lower panel, 40×. (B.1) Bar diagram showing mean values of ikaros positive cells from six individual microscopic fields. (B.2) Bar diagram showing mean percentile of red channel luminosity of ikaros positive cells. *p ≤ 0.001; **p ≤ 0.01, in comparison to iDCs.

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Fig. 5. Expression of activation markers and memory phenotypes on T cells after induction with mDCs. iDC and mDCs were co-cultured with T cell for 48 h. Expression of CD69, CD45RA/CD45RO and CD62L on T cell surfaces were studied after their staining with corresponding antibodies and analysed by flow cytometry. (A) Representative figures from each experiments. (B) Bar diagram shows the expression of these markers on T cells after co-culture with differentiated and matured DCs. Dot plots were presented for CD69/CD8 and CD45RA/RO. Histograms are presented for CD62L. Mean ± SD of six individual experiments are presented in each case. *p < 0.001; **p = 0.0004.

offered restriction of tumor growth, but significantly lesser extent than that group immunized with CEA pulsed BmDCs. Mice of this group also showed disease restricted prolonged survival (Fig. 8B). Similar result was obtained in identical repeat experiment. From an identical set of experiment, splenocytes isolated from all four groups of mice were used for analysis of CEA specific IFN␥ secretion and T cell proliferation. It is apparent from Fig. 8C that maximum IFN␥ was released from T cells of mice immunized with CEA pulsed NLGP-mDC. T cells from this group of mice were proliferated in significantly greater extent following in vitro stimulation with either CEA or ConA (Fig. 8D). 4. Discussion We have previously shown that neem leaf components efficiently stimulate anti-tumor effector T cell functions by upregulating cytotoxic molecules, like, perforin, granzyme B [31] and by secreting cytotoxic cytokines, IFN␥, TNF␣ [32], thereby, creating an immune microenvironment with type 1 commitment [33]. Defective DC maturation in cancer contributes significantly in T cell dysfunctions by imposing poor co-stimulation, downregulated MHC expression and significant upregulation of suppressor molecules, like, indoleamine 2,3-dioxygenase [34]. Correction of defective maturation may be a key for success, as iDCs create type 2 [3] conditions with a cytokine milieu having high TGF␤, IL-10 [35] and promotes the functions of suppressive regulatory T cells [36]. On the otherhand, several reports suggested that matured DCs could prime effectively anti-tumor T cells [4]. In an objective to mature DCs properly, thereby, initiates greater effective T cell response, several DC maturating agents, like, cocktail of proinflammatory cytokines, LPS, TNF␣, IFN␥, Flt3 ligand, CpGODN, were used [13–17]. Cytokine cocktail usually used for ex vivo DC maturation,

promotes expansion of Tregs as well [37]. Other DC maturating agents also show some limitations, either being toxic or expensive. Nontoxic behavior of neem leaf components [38] and several encouraging results with NLGP [21–25,31] prompted us to evaluate its potential as a DC maturating agent. In the present attempt to generate DCs from human blood adherent cells (CD14+ ), standard GMCSF/IL-4 based protocol was followed. To see the effect of NLGP as a DC maturating agent first we checked the purity of NLGP preparation by SE-HPLC. After confirmation of the purity of NLGP, special attention was given to remove the contaminating lymphocytes from monocyte preparation, as T cells produce TNF␣ and CD40L, both known to mature DCs [39]. Our flow cytometric analysis confirms the presence of majority of CD14+ population for DC generation and reduces the possibility of maturation of DCs in T cell dependent manner. Loss of CD14 marker along with high expression of CD1a was noticed during NLGP mediated differentiation. Supplementation of NLGP, as a maturating agent, in iDC culture resulted CD14− CD1alow condition with upregulation of the co-stimulatory molecules, CD80/86, MHC-I and the maturation marker, CD83. Interestingly, with maturation, DC expresses CD1a in most of the cells, but these are CD1alow . This observation was more prominent with NLGP as a maturating agent. NLGP matured DCs differ phenotypically with conventionally matured DC only for CD1a expression, but it express higher or similar levels of CD83 and other co-stimulatory molecules and it also induces strong type 1 response. This observation is in agreement with the observation of Roy et al. [40] for their IL-4 generated DC. Interestingly, all these phenotypic studies clearly suggest the better maturating efficacy of NLGP in comparison to LPS. Some extent of expression of CD83 was noticed in iDC. This may be due to spontaneous maturation of a portion of the DC (spontaneously matured DC) in culture [41]. Around 3% contaminating T cells

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Fig. 6. Regulation of T cell functions under influence of differentiated and matured DCs. MACS purified allogenic and autologous CD8+ CD56− T cells were co-cultured with irradiated iDC, mDC-LPS and mDC-NLGP in ratios of 100:1, 50:1 and 10:1 for either 96 h (proliferation assay) or 72 h (cytokine ELISA) with mitogenic (PHA) stimulation. Data are presented from cultures, where allogenic T cells and DCs were mixed in 10:1 ratios. (A) Proliferation of T cells under different conditions as mentioned was checked by MTT assay. (B) Release of IFN␥ and IL-4 from T cells, cultured in various conditions, was measured by ELISA. # p ≤ 0.0001, *p < 0.0001, **p = 0.0002, + p = 0.08, in comparison to iDC.

in monocyte culture may also participate in expression of CD83 marker on iDCs initially, however, in continuation of the culture these cells may not survive. Interestingly, significant enhancement of the maturation process is only observed after addition of LPS/NLGP. In connection with the generation of human myeloid DCs, NLGP was proved to be effective as a maturating agent of bone marrow (of Swiss mice) derived DCs (BmDC) too and it was emerged superior to LPS in relation to the expression of CD83, MHCs and CD11c. Following co-culture with GMCSF and IL-4 and then maturation with LPS/NLGP for 8 days, the dot blot shows approximately 60% CD11c+ and MHC-II+ cells in mouse system, these values are disparate from the expression of CD1a in human system (≥90%). From our experience and data presented in this communication, it appeared that in human system more numbers of cells appeared CD1a+ positive. From the view of Hackstein et al., IL-4 + GMCSF do not transform all myeloid cells to CD11c+ DC. Only 50–60% cells express CD11c [42]. After phenotypic analysis it is important to evaluate the functional aspects of NLGP matured DCs, and that might be tested by assessing their secretion of IL-12p70, a hallmark of DC function. We have previously demonstrated that NLP is an inducer of IL-12p70 from monocytes/macrophages in CD40/CD40L dependent manner [21]. With enhanced secretion of IL-12p70, NLGP also downregulates release of IL-10, which is required for creation of type 1 immune condition [21,33]. Another important fact should be mentioned here that NLGP-induced release of IL-12p70 from mDCs could not be inhibited by endotoxin inhibitor, polymixin B (50 ␮g/ml) and, thus, ruled out the possibility of endotoxin contamination in this neem preparation. CD8+ DCs in the T cell areas of the spleen have been shown to preferentially activate CD8+ T cells in IL-12 dependent man-

ner, while CD8− DC, which are located in the marginal zone of the spleen, are more efficient in MHC class II presentation and activation of CD4+ T cells [43,44]. These results are consistent with the observations that CD8+ DCs are specialized in cross-presentation of antigens [45]. Our experimental results suggest that NLGP increases the number of both CD8+ and CD8− DC subsets; however, the effect on CD8+ DC was more prominent, thereby, presents antigens to T cells effectively. Moreover, in the signaling cascade involved in DC activation, antigen presentation and induction of optimum type 1 T cell responses, involvement of ikaros is critical [30]. Nuclear ikaros proteins are pleotropic regulators of hematopoiesis and involved in in vitro differentiation of monocytes [46]. Movassagh et al. [30] reported for the first time that ikaros family members play critical role in the functional activation on maturation of DCs. Accordingly, involvement of ikaros proteins in the NLGP-induced maturation of myeloid DCs was studied and we have observed the involvement of ikaros in such DC maturation process. Influence of NLGP-induced optimum maturation of DCs (mDC1) on T cell effector functions was examined. Results obtained from MLRs demonstrated that NLGP matured DCs are efficient to activate protective T cells and induces their proliferation. Upregulation of CD69, CD45RO and downregulation of CD45RA, CD62L confirms T cell activation by NLGP-mDCs than LPS-mDCs. This T cell proliferation was further enhanced by the presence of NLGP in culture, suggesting antigenic behavior of NLGP. NLGP matured DCs induce the secretion of IFN␥ from T cells and decrease the release of IL4. The capacity of mDCs to prime the production of IFN␥ from T cells may be partly attributed to their high surface expression of CD80/CD86 and/or CD40 (data not shown). At the same time NLGP upregulates counterpart molecules on T cell surface, i.e., CD28 and CD40L to promote respective interaction. On the otherhand, it was

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Fig. 7. Induction of tumor cell cytotoxicity by T cells under the influence of differentiated and matured DCs. MACS purified T cells were co-cultured with irradiated iDCs and mDCs for 48 h. mDC-LPS and mDC-NLGP were pulsed with or without CEA before their use in co-culture. (A) Expression of CD8 and CD28 markers on T cells cultured with iDCs and mDCs. (A.1) Representative figures showing CD8 and CD28 expression. (A.2) Bar diagram showing mean values of CD8+ CD28+ cells from six individual experiments. *p ≤ 0.001; **p ≤ 0.0001, in comparison to iDCs. (A.3) Histogram showing the representative expression of CD40L on purified T cells, co-cultured with DCs as mentioned. (B) Cytotoxicity of tumor cells (CEA+ COLO205 and CEA− KB) by autologous (B.1)/allogenic (B.2) T cells in different E:T ratios, when cultured with iDCs and mDCs as mentioned above. *p < 0.001; **p < 0.0001 in comparison to CEA− KB cells. NT, no treatment.

reported by various groups that ligation of CD28 and CD40L promotes induction of IL-4 production in naïve Th cells [47,48]. On contrary, either NLGP matured DCs or mDCs + NLGP enhances the expression of CD28 and CD40L on surface of T cells, thus, promotes opportunity for induction of type 1 immune responses. Optimum maturation of DCs by NLGP is reflected in T cell mediated tumor cell cytotoxicity, when T cells are co-cultured with NLGP matured DCs. NLGP mediated synchronization of complex immune orchestra in DCs and T cells by upregulating HLA-ABC, CD80/86, CD28, CD40/CD40L, etc. possibly responsible for proper antigen presentation along with optimum costimulation and enhanced T cell cytotoxic functions. NLGP matured CEA pulsed DCs present CEA effectively to effector allogenic and autologous T cells, which

is evidenced from enhanced cytotoxicity of CEA+ cells, in comparison to CEA− cells. Confirmation of in vitro cytotoxicity, prompted us to check the potential of NLGP matured CEA pulsed DCs on in vivo growth restriction of CEA+ tumors. Here, we have used BmDCs generated from bone marrows of Swiss mice. Immunization of syngenic mice with NLGP matured CEA pulsed BmDCs always exhibited better result than either LPS matured BmDCs or iDCs in relation to the restriction of the growth of CEA+ mouse colon tumor. NLGP may enhance cross presentation of antigen through MHC-I by stimulating CD8+ DC along with increased costimulation, thus, elicit strong antigen (CEA) specific anti-tumor immunity, which is reflected in in vivo CEA+ tumor growth restriction and increased disease free survival. This tumor growth restriction may

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Fig. 8. Tumor growth restriction, survivability and T cell functions of DC immunized mice. (A) Four groups of Swiss mice (n = 6 in each group) were immunized with iDCs, LPS matured BmDCs, NLGP matured BmDCs and NLGP matured CEA pulsed BmDCs, in alternate weeks, for three times in total. Three days following completion of the immunization, mice were inoculated with mouse colon carcinoma cells (1 × 107 ) subcutaneously on right hind leg quarter. (A) Tumor growth curve, as monitored by caliper measurement using the formula: (width2 × length)/2. Inset: representative mice and isolated tumors from each group. (B) Survivability curve of mice. *p < 0.0001, in comparison to iDC and *p < 0.01 in comparison to mDC-LPS and mDC-NLGP. (C) T cells were cultured in various conditions as mentioned in figure and stimulated with CEA to measure the release of IFN␥ by ELISA. In comparison to, T cell + iDC, # p < 0.0001 and T cell + mDC-LPS + CEA, # p < 0.01. (D) CD8+ T cells were cultured in various conditions as mentioned in figure and stimulated with CEA/ConA for 72 h and proliferation was assessed by MTT colorimetric assay. In comparison to T cell + iDC, + p < 0.0001 and T cell + mDC-LPS/mDC-NLGP, + p < 0.01.

be dependent on activation of CD8+ T cells by NLGP matured DCs, as we observed maximum release of IFN␥ from T cells purified from NLGP-mDC + CEA immunized mice. Maximum CEA specific proliferation was also noted in mice of this particular group. Our data shows that NLGP matured DCs are comparable or to some extent superior to LPS matured DCs. LPS is a potent stimulator of DC maturation [13], however, it is not approved for clinical use because of toxicity concerns [18]. Thus, nontoxic NLGP would obtain clinical importance to replace LPS for the ex vivo generation of DCs. Another popular maturating agent is a commercially available cytokine, TNF␣, which is relatively expensive to produce in large quantities and can have substantial side effects when injected into humans [18,49]. Additionally, cytokine cocktails, those are also used for DC maturation, having prominent regulatory T cell promoting activity [37]. On the otherhand, NLGP is effective to downregulate regulatory T cells in vitro and in vivo (unpublished observation), thus promoting effector T cell activity. Similar to the CpG molecules [50], a major advantage of NLGP is that they are inexpensive to produce, particularly in Indian subcontinent, in large quantities and completely safe to use in humans in any form [38] (human data unpublished). Apart from toxicities, NLGP may be different from LPS in relation to their use of Toll Like Receptor (TLR) pathway. LPS mediates its effect on DC activation via binding to TLR4. Although, downstream signaling responses of NLGP is not explored yet, our preliminary study suggest that NLGP may have specific receptor on various immune cells, like, T cells, monocytes, DCs, etc. as a transmembrane domain (unpublished observation). Signal transduction pathway of

NLGP is currently under investigation. Based on overall data it may be concluded that NLGP may serve as an excellent DC maturating agent and ex vivo generated NLGP matured DCs warrant testing in humans as a potential DC based anti-tumor vaccine strategy. Acknowledgements The authors thank Dr. Jaydip Biswas, Director, CNCI, India for providing necessary facilities. They also thank Dr. Subrata Laskar for his help in characterization of NLGP. Partial grant supports from Indian Council of Medical Research, New Delhi (Grant No. Immuno/18/11/08/2006-ECD-I) and University Grant Commission, New Delhi (Grant No. F.2-3/2000 (SA-1) to A.B.) are acknowledged. References [1] Liu YJ, Holger K, Vassili S, Gilliet M. Dendritic cell lineage, plasticity and cross regulation. Nat Immunol 2001;2(7):585–9. [2] Josien R, Li HL, Ingulli E, Sarma S, Wong BR, Vologodskaia M, et al. TRANCE, a tumor necrosis factor family member, enhances the longevity and adjuvant properties of dendritic cells in vivo. J Exp Med 2000;191(3):495–502. [3] Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, et al. Immunobiology of dendritic cells. Annu Rev Immunol 2000;18:767–811. [4] Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998;392(6673):245–52. [5] Cellia M, Saliustro F, Lanzavecchia A. Origin, maturation and antigen presenting function of dendritic cells. Curr Opin Immunol 1997;9(1):10–6. [6] Brittingham KC, Ruthel G, Panchal RG, Fuller CL, Ribot WJ, Hoover TA, et al. Dendritic cells endocytose Bacillus anthracis spores: implications for anthrax pathogenesis. J Immunol 2005;174(9):5545–52.

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