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

Abstract

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.

Fig. 1.

Comparative status of GM-CSF- and IL-4-differentiated iDCs from myeloid cells obtained from CaCx patients and healthy individuals. Monocytes were isolated from blood specimens obtained from CaCx patients (n = 6) and healthy individuals (n = 6) and differentiated with GM-CSF plus IL-4 for 5 days. The statuses of CD1a and CD14 in iDCs before maturation between CaCx patients and healthy individuals were compared. (A) Forward scatter (FSC)/side scatter (SSC) dot plot of DCs; (B, D) acquisition of CD1a phenotypes, depicted by a representative figure (B) and a bar diagram showing the percentage of positive cells (D); (C, E) loss of CD14 phenotypes, depicted by a representative figure (C) and a bar diagram showing the percentage of positive cells (E). *, P < 0.01 (CD1a cells in comparison to healthy cells); **, P < 0.001 (CD14 cells in comparison to healthy cells).

Fig. 2.

Expression of costimulatory and maturation markers on dendritic cells (DCs) obtained from CaCx patients and healthy individuals. Human myeloid cells from CaCx patient (n = 6) and healthy control (n = 6) samples were differentiated with GM-CSF/IL-4 and matured with either LPS to DCs (mDCLPS) or NLGP to DCs (mDCNLGP). Expression of CD1a (A), CD40 (B), CD83 (C), CD80 (D), CD86 (E), and HLA-ABC (F) was studied using various fluorescence-labeled markers as indicated and analyzed by flow cytometry using Cell Quest and WINMD1 software. An appropriate isotype-matched antibody was used as a negative control. The percentage of positive cells was monitored by flow cytometric analysis after gating of DC-rich zones from FSC/SSC plots. A representative figure from each case is presented. Bar diagrams show the average expression levels as the mean percentages of positive cells ± SDs. The values indicated on the histograms are the mean fluorescence intensities (MFI) of the positive cells in the gated population. The results are representative of six similar experiments. Levels of significance are as follows. (A) For CD1a, iDCs versus NLGP DCs from healthy individuals, P < 0.05; iDCs from healthy individuals versus iDCs from CaCx patients, P = 0.0047. (B) For CD40, iDCs versus NLGP/LPS DCs from healthy individuals, P < 0.001; iDCs versus NLGP/LPS DCs from CaCx patients, P < 0.005; iDCs from healthy individuals versus iDCs from CaCx patients, P = 0.002. (C) For CD83, iDCs versus NLGP/LPS DCs from healthy individuals and CaCx patients, P < 0.001; iDCs versus LPS DCs from CaCx patients, P < 0.01; iDCs from healthy individuals versus iDCs from CaCx patients, P = 0.0072. (D) For CD80, iDCs versus NLGP/LPS DCs from healthy individuals, P < 0.001; iDCs versus NLGP/LPS DCs from CaCx patients, P < 0.001. (E) For CD86, iDCs versus NLGP/LPS DCs from healthy individuals, P < 0.001. (F) For HLA-ABC, iDCs versus NLGP/LPS DCs from healthy individuals, P < 0.01; iDCs versus NLGP DCs from CaCx patients, P = 0.0015; iDCs from healthy individuals versus iDCs from CaCx patients, P = 0.03.

Fig. 3.

Type 1 polarization by NLGP-matured DCs. Immature DCs generated from monocytes obtained from CaCx patients (n = 6) and healthy individuals (n = 6) were matured in vitro with NLGP/LPS, and the secretory statuses of type 1 cytokine IL-12 (A) and type 2 cytokine IL-10 (B) were assessed in culture supernatants by ELISA. The mean ± SD is presented in each case. *, P < 0.001 (for iDCs versus NLGP/LPS DCs from healthy individuals and for iDCs versus NLGP/LPS DCs from CaCx patients).

Fig. 4.

Secretion of IFN-γ from autologous T cells induced by NLGP-matured DCs. MACS-purified autologous CD8⁺ T cells obtained from CaCx patients and healthy individuals (n = 6 in each case) were cocultured with iDCs, mDCs-LPS and mDCs-NLGP at ratios of 10:1 for 48 h. Intracellular release of IFN-γ in T cells, cultured under various conditions, was measured using fluorescence-labeled anti-IFN-γ antibody and analyzed by flow cytometry using Cell Quest software. An appropriate isotype-matched antibody was used as a negative control. The percentage of positive cells was monitored by flow cytometric analysis after gating of the lymphocyte-rich zone. (A) A representative dot plot from each case is shown. (B) The bar diagram shows the average expression as the percentage of positive cells.*, P < 0.05 (iDCs versus LPS DCs from healthy individuals/CaCx patients); **, P < 0.01 (iDCs versus NLGP DCs from healthy individuals/CaCx patients).

Fig. 5.

Induction of tumor cell cytotoxicity by antigen-primed T cells. MACS-purified T cells obtained from CaCx patients (n = 6) and healthy individuals (n = 6) were cocultured with irradiated iDCs and mDCs for 48 h. mDCs-LPS and mDCs-NLGP were pulsed with HeLa cell extract before use in coculture. CTLs generated under these conditions were used to lyse antigen-positive HeLa cells and antigen-negative U937 cells after coculturing at an effector/target (E/T) ratio of 1:10. Bar diagrams represent the mean cytotoxicity values ± SDs obtained from six individual experiments. *, P < 0.001 (iDCs versus NLGP/LPS DCs from healthy individuals/CaCx patients).

Fig. 6.

Induction of allogeneic T cell proliferation after antigen presentation by DCs. Allogeneic CD8⁺ T cells obtained from CaCx patients and healthy individuals (n = 6 in each case) were purified by MACS analysis and cocultured with iDCs, mDCs-LPS, and mDCs-NLGP pulsed with HeLa extract at a ratio of 10:1 for 96 h. Proliferation was checked by the MTT assay. The bar diagram represents the averages from six separate experiments. *, P < 0.001 (iDCs versus NLGP/LPS DCs from healthy individuals/CaCx patients).