Immunodominant T cell epitopes in different adjuvants vaccination mice.

Spleens were collected from mice on day 10 post vaccination with antigen UreB incorporated with adjuvants CpG, MDP and MPLA respectively. Then, these cells were cultured in vitro and stimulated with a panel of overlapping UreB 18mer peptides to assess the responsiveness of CD4+ T cells by ICS for IFN-γ. The percentages of CD4+ T cells secreting IFN-γ against each peptide were determined by flow cytometry and shown. Locations of dominant peptides in different groups are indicated.

MHC-II peptidome and proteome measurements in adjuvants treated APCs.

A20 cells were treated with CpG, MDP and MPLA incorporated with H. pylori antigens for 12h, respectively. Then, most of the cells (108) were lysed for immunopeptidomics, and the remaining cells (107) were used for proteomics. (A) Flow chart of the experiment. (B) The number of MHC peptides identified in different adjuvants treated groups. n=3. Numbers indicate the Mean value. (C) Length distribution of MHC peptides in different adjuvants treated groups. (D) Sequence motifs of MHC peptides identified in adjuvants treated groups. (E) Binding heatmaps of all eluted MHC peptides between 9-22mers in adjuvants-treated groups were predicted and assigned to alleles using NetMHCIIpan. ns: no significant difference (P>0.05).

Profiling exogenous MHC-II peptides in adjuvants treated APCs.

(A) Peptide locations across the H. pylori genome from MHC-II immunopeptidomes. (B) Rank plot of each protein abundance detected in the whole proteome of bacterial ultrasonic supernatant antigens. Proteins identified in immunopeptidomes are annotated with their respective gene names. (C) MHC-II presentation potential of bacterial proteins. All reported H. pylori proteins were ranked according to the ratio between the number of peptides predicted to be presented by MHC-II alleles (rank≤2) and the total number of 13- to 17-mers. Proteins identified in immunopeptidomes are annotated with their respective gene names. (D) Heatmap of exogenous MHC peptides from different adjuvant groups. The identified sequences are shown. (E) Numbers of MHC peptides derived from bacteria and host were compared among different adjuvant groups. n=3. **p < 0.01, ****P < 0.0001.

Antigen phagocytosis of APCs treated with different adjuvants.

(A) Comparison of bacterial protein abundance in APCs 12h post adjuvants stimulation from whole proteomes. (B) Abundances of bacterial proteins from immunopeptidome and proteome were compared among adjuvant groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****P < 0.0001.

The effects of adjuvants on antigen presentation.

(A) Rank plot of protein abundances from host and bacteria in the whole-proteome of different adjuvant groups. Bacterial proteins are marked with red, and some of them are annotated with their respective gene names. (B) Similar to (A) but for MHC peptide abundances from immunopeptidomes of different adjuvant groups. Bacterial MHC peptides are annotated with their respective amino acid sequence. (C) Volcano plots comparing protein levels between PBS and adjuvants-treated groups in the whole-proteome. Proteins involved in antigen processing, ubiquitination, proteasome & peptidase, and IFN pathways are colored accordingly. Above the dashed line (p-value < 0.01) means significant.

Binding affinity of MS-detected peptides were determined.

(A) IC50 values of the presented and deficient peptides post adjuvants stimulation from immunopeptidome binding to H2-IA and H2-IE were predicted by the NN align method using the IEDB website. A high IC50 value means a low binding stability. (B) Distribution of proteins corresponding to bacterial MHC peptides from immunopeptidome. The numbers of peptides identified by MS for each protein are indicated. (C) Information of 10 synthetic peptides from Top4 presented and deficient proteins. ×: Presence of peptides in the corresponding group. -: Peptides missing in the corresponding group. (D) Mirror plots with fragment ion mass spectra to confirm the sequences of MHC peptides from immunopeptidome. Positive y-axis, MHC-II IP sequences; negative y-axis, synthetic peptides. (E) Competitive binding curve of synthetic peptides for MHC II H2-IA allele. The binding curves of peptides presented in adjuvant groups are marked with red.

T cell responses induced by MS-detected peptides with different binding stability were analyzed.

Five BALB/c mice were immunized with a pool of 10 synthetic peptides. On day 10 and day 28 post immunization, splenocytes were isolated and stimulated with individual peptides for IFN-γ Elisopt assay. (A) Flow chart of the experiment. (B) and (C) Elispot results on day 10 and day 28. OVA peptide and non-stimulated wells were used as a negative control. PMA stimulation was used as a positive control. Responses against peptides presented in adjuvant groups are marked with red. The dashed line is the 3× median of the OVA peptide used as the threshold for positive responses. The box shows the quartiles, the bar indicates median, and the whiskers show the distribution. Elispot images of positive responses from one of the immunized mice are shown. Numbers indicate the spot counts. (D) Epitope-specific CD4+ T cells from spleens of immunized mice were expanded in vitro and the IFN-γ producing CD4+ T cells were assessed using the peptides pool (left). Then, the presented peptide recA#23 and the deficient peptides ureA#2 and ureA#3 post adjuvant treatment were titrated to restimulate the expanded cells (right). IFN-γ responses of CD4+ T cells were detected by FACS. The responses induced by the indicated peptides at 50μM were considered as 100%. And all the other responses were evaluated by their relative strength.