HLA-DR and HLA-DP restricted epitopes from human cytomegalovirus glycoprotein B recognized by CD4+ T-cell clones from chronically infected individuals

Abstract

Purpose: Helper CD4(+) T cells presumably play a major role in controlling cytomegalovirus (CMV) by providing help to specific B and CD8(+) cytotoxic T cells, as well as through cytotoxicity-mediated mechanisms. Since CMV glycoprotein B (gB) is a major candidate for a subunit vaccine against CMV, we searched for gB-epitopes presented by human leukocyte antigen (HLA)-class II molecules.

Methods: Dendritic cells obtained from CMV-seropositive donors were loaded with a recombinant gB and co-cultured with autologous CD4(+) T cells. Microcultures that specifically recognized gB were cloned by limiting dilution using autologous Epstein-Barr virus (EBV)-immortalized B cells pulsed with gB as antigen-presenting cells. To pinpoint precisely the region encoding the natural epitope recognized by a given CD4(+) clone, we assessed the recognition of recombinant Escherichia coli expressing gB-overlapping polypeptides after their processing by autologous EBV-B cells.

Results: We isolated several gB-specific CD4(+) T-cell clones directed against peptides gB(190-204), gB(396-410), gB(22-36) and gB(598-617) presented by HLA-DR7, HLA-DP10 and HLA-DP2. While their precise role in controlling CMV infection remains to be established, gB-specific CD4(+) T cells are likely to act by directly targeting infected HLA-class II cells in vivo, as suggested by their recognition of EBV-B cells infected by the Towne CMV strain.

Conclusions: The characterization of such gB-epitopes presented by HLA-class II should help to understand the contribution of CD4(+) T-cell responses to CMV and may be of importance both in designing a vaccine against CMV infection and in immunomonitoring of subjects immunized with recombinant gB or with vectors encoding gB.

Fig. 1

Overview of the procedure used to obtain and characterize anti-gB CD4⁺ T-cell clones

Fig. 2

Characteristics of clone 1A10. a Recognition of EBV-B cells loaded with recombinant gB-expressing bacteria by CD4⁺ T-cell clone 1A10. CD4⁺ T-cell clone 1A10 (3000 cells) were co-cultured for 20 h with autologous CMV001 EBV-B cells (20 000 cells/well) loaded or not with gB (10 μg/ml) or with recombinant bacteria that expressed each of the 4 overlapping gB constructs (A, B, C, D) or each of five overlapping constructs spanning the A (A1 to A5), B (B1 to B5), C (C1 to C5) or D (D1 to D5) fragments. The amount of IFN-γ secreted in the supernatant of the co-cultures was measured using standard ELISA. The results shown represent an average of duplicate co-cultures. b Recognition of gB overlapping peptides by CD4⁺ T-cell clone 1A10. EBV-B cells were incubated for 2 h with each of the gB overlapping peptides (1 μg/ml). CD4⁺ T-cell clone 1A10 (3000 cells) were then co-cultured with autologous peptide-pulsed EBV-B cells (20 000 cells) for 18 h. IFN-γ production in the supernatant was measured by ELISA. The results shown represent the average of triplicate cultures. c Identification of the minimal gB epitope recognized by CD4⁺ T-cell clone 1A10. EBV-B cells were distributed in microwells (2 × 10⁴ cells) and incubated for 2 h with various concentrations of each individual peptide. A total of 3 to 5 × 10³ cells from autologous CD4⁺ T-cell clone 1A10 was added and the presence of IFN-γ production in the supernatant was measured by ELISA after overnight culture. The results shown represent an average of duplicate cocultures. d Inhibition of the gB peptide recognition by anti-HLA-DP, -DQ or -DR Abs. Autologous EBV-B cells were incubated with 10 μg/ml of recombinant gB for 20 h. CD4 T-cell clones (3 000 cells) were then co-cultured with 20 000 protein-loaded EBV-B cells for 20 h. Inhibition with anti-HLA class II mAbs was performed by addition of 10 μg/ml of mAb during the co-culture. IFN-γ production was measured by ELISA after 18 h. e HLA-class II restriction analysis of the gB epitope. The gB-specific CD4⁺ T-cell clone 1A10 was exposed to gB-loaded autologous or allogeneic EBV-B cells. IFN-γ production was measured by ELISA after overnight co-culture. The results shown represent the average of triplicate co-cultures

Fig. 3

Characteristics of clone 2G12. The procedure used for clone 2G12 was the same as the one described in Fig. 2. a Recognition of EBV-B cells loaded with recombinant gB-expressing bacteria by CD4⁺ T-cell clone 2G12. b Recognition of gB overlapping peptides by CD4⁺ T-cell clone 2G12. c Identification of the minimal gB epitopes recognized by CD4⁺ T-cell clone 2G12. d Inhibition of the gB peptide recognition by anti-HLA-DP, -DQ or -DR Abs. e HLA-class II restriction analysis of the gB epitope recognized by CD4⁺ T-cell clone 2G12

Fig. 4

Characteristics of clone 2C4. The procedure used for clone 2C4 was the same as the one described in Figs. 2 and 3. a Recognition of EBV-B cells loaded with recombinant gB-expressing bacteria by CD4⁺ T-cell clone 2C4. b Recognition of gB overlapping peptides by CD4⁺ T-cell clone 2C4. c Identification of the minimal gB epitope recognized by by CD4⁺ T-cell clone 2C4. d Inhibition of the gB peptide recognition by anti-HLA-DP, -DQ or -DR Abs

Fig. 5

Characteristics of clone B3A5. The procedure used for clone B3A5 was the same as the one described in Figs. 2 and 3. a Recognition of EBV-B cells loaded with recombinant gB-expressing bacteria by CD4⁺ T-cell clone B3A5. b Inhibition of the gB peptide recognition by anti-HLA-DP, -DQ or -DR Abs. c Identification of the minimal gB epitopes recognized by by CD4⁺ T-cell clone B3A5. d HLA-class II restriction analysis of the gB epitope recognized by CD4⁺ T-cell clone B3A5

Fig. 6

Recognition of HCMV-infected cells by two CD4⁺ T-cell clones directed against gB and expression of GrB by clone B3A5. gB-specific CD4⁺ T-cell clones B3A5 (a) or 1A10 (b) recognize autologous EBV-B cells infected with the Towne laboratory strain. CD4⁺ T-cell clones were stimulated with autologous EBV-B cells, loaded or not either with a recombinant gB protein or with the relevant gB-derived peptide, or infected with the Towne CMV strain (10 pfu/cell). IFN-γ production in the supernatant was measured by ELISA after 36 h. (c) Anti-GrB labeling of clone B3A5. Percentages of CD3⁺CD4⁺ or CD4⁺GrB⁺ double positive cells are indicated in the upper right corner of each subpanel