New T cell epitopes identified from an anti-idiotypic antibody mimicking ovarian cancer associated antigen

Abstract

Anti-idiotype (Id) antibodies can be used to induce specific cellular immune responses against tumor antigens, but the mechanism of antigenicity is not always clear. We previously reported an anti-Id antibody, 6B11, which mimics human ovarian cancer associated antigen OC166-9. To explore the molecular basis of cellular immune response induced by 6B11, a panel of peptides derived from complementarity determining region (CDR) of 6B11 were synthesized. After a series of immunologic experiments, we found that the light chain CDR3 peptide and heavy chain CDR3 peptide were the MHC class I and class II epitopes of 6B11, respectively. The combination of MHC class I and class II epitopes is more effective than 6B11 in inducing specific cellular immune response against ovarian cancer. Our study provided the structural basis of antigenicity of 6B11. The identification of antigen-specific T cell eptitopes in 6B11 should facilitate the design of epitope-based vaccine against human ovarian cancer.

Fig. 1

Confocal laser scanning microscopy and FACS detects the MHC class I molecule surface expression of T2 cells. T2 cells were incubated with 30 μg/ml of each peptide for 24 h respectively. The surface expressions of HLA-A*0201 molecules at T2 cells were recorded using indirect antibody staining and confocal analysis. a high affinity peptide, b intermediate affinity peptide, c low affinity peptide, d zero affinity peptide, e single T2 cell control. The ability of peptides to stabilize HLA-A*2 on the surface of T2 cells was assessed by FACS after staining peptide-treated cells with anti-HLA-A*2 antibody. Data are showed as MFI f

Fig. 2

Phenotypic analysis of mature DCs and detection of the 6B11 CDRs peptides responsible for inducing proliferative response of 6B11-primed T cell. a–d Immature DCs were matured with rh IFN-α , rh TNF-α, rh IL-6 and PGE2 for 24 h. Expression levels of several cell surface markers on mature DCs were determined by FACS analysis. Data are representative of one experiment out of four performed. e 6B11-CTLs were in vitro stimulated with PHA, 6B11 or peptide-pulsed DCs and unpulsed DCs preincubated with anti-MHC class II antibody or not at a ratio of 10:1 for 120 h, and the proliferative response was measured using BrdU ELISA. f CD4 T cells of 6B11-CTLs were separated by magnetic beads sorting and used as responder cells for cell proliferation assay

Fig. 3

Cytotoxicity of 6B11 CDRs deriving peptides-specific CTLs. Lymphocytes from HLA-A*2 donors were cocultured with 6B11 or peptide and KLH-pulsed DCs at a ratio of 10:1 for 3 cycles. a–j The cytotoxic activities of each peptide and KLH-primed CTLs against this peptide loaded T2 cells (column-dotted), HOC1A (column-grey shading), HLE (column-no fill) or corresponding targets preincubated with anti-MHC class I antibody (column-diagonal striped, column-horizontal striped, column-netted designed) were tested at different E:T ratios. The cytotoxic activities of each peptide and KLH-primed CTLs against unloaded T2 cells (column-grey shading) and unpulsed DCs-primed T cells (column striped) or naïve T cells (column black fill) against each peptide loaded T2 cells were also tested as negative controls. k The cytotoxic activities of VH or (and) VL CDR3 peptide-CTLs and 6B11-CTLs against HOC1A, HLE or K562 were tested at different E:T ratios. The cytotoxic activities of unpulsed DCs-primed T cells (column-grey shading)and naïve T cells (column diamond designed) against HOC1A were also tested as negative controls

Fig. 4

Comparison of the cytokines profile induced by 6B11 and identified peptides. 6B11, VH CDR3 peptide, VL CDR3 peptide or VH and VL CDR3 peptides-specific CTLs were cocultured with HOC1A and HLE cells for 48 h. The unpulsed DCs-primed T cells and naïve T cells were used as negative controls. The supernatants were harvested to detect the production of IL-2, IFN-γ and IL-4 with sandwich ELISA

Fig. 5

Similar frequency of IFN-γ producing cells presents in 6B11-CTLs and CDR3 peptides-primed CTLs. VH and VL CDR3 peptides specific CTLs, CD4 or CD8 CTLs components were cocultured with HOC1A and HLE cells at a ratio of 20:1 for 24 h, then all cells were discarded. The forming spots representing the IFN-γ producing cells were counted. 6B11-CTLs and unpulsed DCs-primed T cells were used as positive and negative controls respectively