BCL1 lymphoma protection induced by idiotype DNA vaccination is entirely dependent on anti-idiotypic antibodies

Abstract

DNA vaccination with the idiotype (Id) of tumour B-cell membrane immunoglobulins (Ig) is a validated strategy to induce tumour protection to several mouse lymphomas. The relative contribution of anti-Id antibodies and T lymphocytes to tumour rejection is still debated. Previous studies in the BCL1 lymphoma model showed that scFv DNA immunisation induces a polyclonal antibody response restricted to conformational epitopes formed by the parental V(L)/V(H) association. We implemented a system based on this specificity to investigate the mechanism of BCL1 lymphoma protection induced by DNA immunisation. Antibody response and survival of mice immunised with the tumour Id scFv were compared with those of mice immunised simultaneously with two chimeric scFvs, containing either the tumour-derived V(L) or V(H) paired to an irrelevant V(H) or V(L) domain, respectively. Animals vaccinated with one or both chimeric constructs were not protected, despite the exposure to all putative tumour Id-derived MHC class I and class II T-cell epitopes. In addition, conformational antibodies induced by DNA vaccination caused tumour cells apoptosis and cell cycle arrest in vitro and transferred protection in vivo. Therefore, lymphoma rejection appears to be completely dependent on the induction of anti-Id antibodies.

Fig. 1

Proliferation of splenic CD4⁺ T cells. DCs were purified from spleens of BALB/c control mice and pulsed with 1.5 μg/ml of hIgG, Id^BCL1/CH3 and Id^BCL1. DCs were then incubated with CD4⁺ T cells extracted from mice immunised with pCH3 or pBCL1. T-cell proliferation was detected by ³H-thymidine incorporation.

Fig. 2

a DNA-immunising constructs. The construct pBCL1 contains both BCL1-derived V_L and V_H domains, while the p6C6 vector contains the Id of an irrelevant murine monoclonal antibody (mAb 6C6). The chimeric constructs pchB and pchC contain the V_L^BCL1/V_H^6C6 and V_L^6C6/V_H^BCL1 domains, respectively. b Strategy of immunisation. Eight mice/group were immunised four times at 0, 14, 28, and 42 days with the indicated constructs. Each group was exposed to the indicated combinations of putative MHC class I and II T-cell epitopes derived from the V_L^BCL1 and/or V_H^BCL1 domains.

Fig. 3

Antibody responses induced by DNA vaccination. a Ab levels were measured by ELISA against γ1-CH3 or Id^BCL1 on blood samples collected after three DNA shots at 1:200 dilutions. Reactivity is plotted as mean for each mice group and the standard deviations (SDs) among mice sera are indicated. b Mice sera were diluted 1:200 and analysed by flow cytometry on BCL1.3B3 cells. The reactivity of one mouse per group is shown as a representative response. c Ab reactivities of sera from mice immunised with one or both chimeric constructs were analysed by flow cytometry. Sera were incubated at 1:200 dilution with Sp2/0 cells displaying on their surface the indicated Ids. Sera of nonimmunised mice were used as negative controls. The reactivity of one mouse per group is shown as a representative response.

Fig. 4

Survival of immunised mice following challenge with BCL1 lymphoma. Two weeks after the fourth shot, mice received 5×10⁴ viable BCL1 lymphoma cells intraperitoneally. Animals were then followed up until death. The p value refers to the pBCL1 group compared with the p6C6 control group.

Fig. 5

Immune sera transfer. Mice were injected intraperitoneally with 5×10⁴ viable BCL1 lymphoma cells and 10 or 100 μl of control or pBCL1-induced sera. Animals were followed up for survival. p Values refer to pBCL1 groups compared to control group.

Fig. 6

a Dose/effect response on BCL1.3B3 cells treated with different amounts of nonimmune or pBCL1-induced sera. Cell death was detected using annexin V and PI. Percentage of viable cells is indicated. A20 B-cell line was used as negative control. Data are normalised to the negative control (nonimmune/BCL1.3B3), plotted as 100%. b Induction of BCL1 tumour cell death following o/n incubation with pBCL1 and pchB+pchC induced sera or anti-FAS (100 ng) antibody. Data are normalised to the negative control (untreated cells), plotted as 100%. SDs of three different experiments are indicated. c Tumour cells were treated with a nonimmune and a pBCL1-induced serum and stained with annexin V. Viable cells were gated and analysed for cell cycle status with PI. The percentages of viable cells on S/G2/M phases (M1 region) are indicated. d Amplification of BCL1 V_H in surviving mice. RT-PCR was performed on 10⁴ splenocytes of control (lane 1) and surviving (lanes 2, 3 and 4) mice, and on 10² (lane 5) and 10³ (lane 6) splenocytes from tumour-bearing mice. Amplification products were separated on a 2% agarose gel.