Salmonella enterica serovar Typhimurium immunotherapy for B-cell lymphoma induces broad anti-tumour immunity with therapeutic effect

Abstract

Despite the efficacy of current immune-chemotherapy for treatment of B-cell non-Hodgkin lymphoma, a substantial proportion of patients relapse, highlighting the need for new therapeutic modalities. The use of live microorganisms to develop anti-tumoural therapies has evolved since Coley's toxin and is now receiving renewed attention. Salmonella Typhimurium has been shown to be highly effective as an anti-tumour agent in many solid cancer models, but it has not been used in haemato-oncology. Here, we report that intra-tumoural administration of LVR01 (attenuated S. Typhimurium strain with safety profile) elicits local and systemic anti-tumour immunity, resulting in extended survival in a lymphoma model. LVR01 induces intra-tumoural recruitment of neutrophils and activated CD8(+) T cells, as well as increasing the natural killer cell activation status. Furthermore, a systemic specific anti-tumour response with a clear T helper type 1 profile was observed. This approach is an alternative therapeutic strategy for lymphoma patients that could be easily moved into clinical trials.

Keywords: bacteria; tumour immunology; vaccination.

Figure 1

In vitro cytotoxicity of Salmonella Typhimurium LVR01 in A20 cells. Infection assays were carried out at a MOI 100 : 1 for 1 hr, and cell viability was determined by MTT and Annexin V – Propidium Iodide (PI) flow cytometry assays. (a) Plot of MTT absorbance of the replicates (n = 10). (b) MTT assay. Absorbance at 570 nm (mean ± standard deviation) and viability (%) (P = 0·002, Student's t-test). (c) Percentage of apoptotic and necrotic cells (mean ± standard deviation) determined by Annexin V-PI, *P < 0·05.

Figure 2

Biodistribution of Salmonella LVR01 in A20 tumour-bearing mice. Mice were inoculated subcutaneously with 1 × 10⁶ A20 cells at day −20, and at day 0 mice received 1 × 10⁶ colony-forming units (CFU) bacteria intra-tumourally. Mice were sacrified at days 1, 5, 9 and 25 after Salmonella inoculation and the number of bacteria in tumour, spleen and liver was determined. Graphs show mean of CFU per gram of tissue (n = 3).

Figure 3

Mouse survival and tumour volume curve post-tumour challenge. Mice were challenged (subcutaneously) with 1 × 10⁶ A20 lymphoma cells, and when tumour became palpable were divided in groups and treated intra-tumourally with one of the following: 3-LVR01, 1-LVR01 or PBS. n = 10 mice/group. (a) Kaplan–Meier plot (log rank, P = 0·0001). (b) Tumour volume curve.

Figure 4

Percentages of (a) CD4⁺ T cells, CD8⁺ T cells, natural killer (NK) cells, (b) neutrophils, and (c) activated cells infiltrating tumour cells. Fine needle aspirates (FNA) were taken from tumours at day 38 post-tumour cell inoculation (p.t.i.) and then prepared as a single-cell suspension for flow cytometry analysis. Data are shown as mean ± standard deviation. n = 10 per group. *P < 0·05 compared with PBS group.

Figure 5

Tumour cytokine gene expression. The expression of cytokine genes was assessed by quantitative RT-PCR on total tumour RNA at day 40 post-tumour cell inoculation (p.t.i.). Values were normalized to that of β-actin mRNA, and the results are expressed relative to mRNA levels in the PBS group. Data are shown as mean ± standard deviation. n = 5 per group. *P < 0·05 compared with PBS group.

Figure 6

Splenocytes antigen-specific cytokine responses in the different groups of mice. Cytokine concentrations were measured by cytometric bead array on the supernatant of splenocyte cultures after 4 days of stimulation. Data shown are (a) interferon-γ (IFN-γ) and (b) interleukin-12p70 (IL-12p70) concentration in cell cultures stimulated with irradiated A20 cells (anova, P = 0·0001 and P = 0·0025). Data shown as mean ± standard error. n = 5 per group. *P < 0·05 compared with PBS group.

Figure 7

Humoral responses against A20 lymphoma cells. Seric antibody levels against A20 lymphoma cell homogenate were determined by ELISA. Data are shown as mean ± standard deviation. n = 5 per group. *P < 0·05 compared with PBS group.