Neem Leaf Glycoprotein Activates CD8+ T Cells to Promote Therapeutic Anti-Tumor Immunity Inhibiting the Growth of Mouse Sarcoma

Abstract

In spite of sufficient data on Neem Leaf Glycoprotein (NLGP) as a prophylactic vaccine, little knowledge currently exists to support the use of NLGP as a therapeutic vaccine. Treatment of mice bearing established sarcomas with NLGP (25 µg/mice/week subcutaneously for 4 weeks) resulted in tumor regression or dormancy (Tumor free/Regressor, 13/24 (NLGP), 4/24 (PBS)). Evaluation of CD8+ T cell status in blood, spleen, TDLN, VDLN and tumor revealed increase in cellular number. Elevated expression of CD69, CD44 and Ki67 on CD8+ T cells revealed their state of activation and proliferation by NLGP. Depletion of CD8+ T cells in mice at the time of NLGP treatment resulted in partial termination of tumor regression. An expansion of CXCR3+ and CCR5+ T cells was observed in the TDLN and tumor, along with their corresponding ligands. NLGP treatment enhances type 1 polarized T-bet expressing T cells with downregulation of GATA3. Treg cell population was almost unchanged. However, T∶Treg ratios significantly increased with NLGP. Enhanced secretion/expression of IFNγ was noted after NLGP therapy. In vitro culture of T cells with IL-2 and sarcoma antigen resulted in significant enhancement in cytotoxic efficacy. Consistently higher expression of CD107a was also observed in CD8+ T cells from tumors. Reinoculation of sarcoma cells in tumor regressed NLGP-treated mice maintained tumor free status in majority. This is correlated with the increment of CD44hiCD62Lhi central memory T cells. Collectively, these findings support a paradigm in which NLGP dynamically orchestrates the activation, expansion, and recruitment of CD8+ T cells into established tumors to operate significant tumor cell lysis.

Absence of CD4+ T cell help generates corrupt CD8+ effector T cells in sarcoma-bearing Swiss mice treated with NLGP vaccine

“…Neem leaf preparation was then extensively dialyzed against PBS and concentrated. Glycoprotein present in this preparation (NLGP) was isolated and characterized by the method described before [18,26]. The NLGP (25 g/100 l/mice) was injected to the mice using the insulin syringe (needle size: 0.30 mm to 8 mm).…”

“…T cell surface phenotypic markers were assessed flow cytometrically after labeling of cells (1 × 10 6 ) with anti-mouse fluorescence labeled antibodies (CD4, CD8, CD44, CD69, KLRG1, CD28, and CD40) as described before [18,19]. Similarly, intracellular molecules (IFN␥, Granzyme B, Runx3, and pStat5) were stained with similar antibodies using cytofix-cytoperm reagents as discussed earlier [19,21].…”

“…Accordingly, NLGP-vaccine model was selected where tumor growth restriction is strictly dependent on CD8 + effector T cells [18][19][20][21]. The effect of CD4 + helper T cell depletion on murine sarcoma growth, under NLGP vaccine was compared.…”

“…In recent times, a neem leaf glycoprotein (NLGP)-vaccine model has been developed, where NLGP-mediated therapeutic tumor growth restriction is CD8 + T cell dependent [18][19][20][21]. NLGP enhances perforin/granzyme B expression in CD8 + T cells [19], occasionally downregulated in tumor microenvironment.…”

Azadirachta indica

“…This NLGP-generated anti-CEA immune response would be effective as a vaccine to lyse CEA(+) tumours in vitro and in vivo. Recent studies by Mallick et al ( 2013a ) found that treatment of mice bearing established sarcomas with neem leaf glycoprotein (NLP) (25 μg/mice/week subcutaneously for 4 weeks) resulted in tumour regression or dormancy. NLGP promoted the therapeutic antitumour immunity in mice by dynamically modulating the activation, expansion and recruitment of CD8(+) T cells into established tumours to induce signifi cant tumour cell lysis and inhibit growth of mouse sarcoma.…”