Volume 144, Issue 3, Pages 591-600 (March 2013) A Dominant CD4+ T-Cell Response to Helicobacter pylori Reduces Risk for Gastric Disease in Humans  Li Chen, Bin Li, Wu–Chen Yang, Jia–Lin He, Ning–Yi Li, Jian Hu, Ya–Fei He, Shu Yu, Zhuo Zhao, Ping Luo, Jin–Yong Zhang, Hai–Bo Li, Ming Zeng, Dong–Shui Lu, Bo–Sheng Li, Hong Guo, Shi–Ming Yang, Gang Guo, Xu–Hu Mao, Weisan Chen, Chao Wu, Quan–Ming Zou  Gastroenterology  Volume 144, Issue 3, Pages 591-600 (March 2013) DOI: 10.1053/j.gastro.2012.12.002 Copyright © 2013 AGA Institute Terms and Conditions

Figure 1 HpaA-specific CD4+ T cell responses in H pylori–infected individuals. PBMCs from (A) H pylori–infected subject 5751 or (B) H pylori–uninfected subject 5248 were stimulated with recombinant HpaA, and IFN-γ–producing CD4+ T cells were assessed using the HpaA peptide pool on day 13. (C) PBMCs from 34 H pylori infection–negative (–) and 40 H pylori infection–positive (+) subjects were stimulated with recombinant HpaA, and IFN-γ–producing CD4+ T cells were assessed as previously mentioned. Gastroenterology 2013 144, 591-600DOI: (10.1053/j.gastro.2012.12.002) Copyright © 2013 AGA Institute Terms and Conditions

Figure 2 Mapping immunodominant HpaA epitopes. The same HpaA-specific T-cell lines used in Figure 1 derived from 5 H pylori–infected subjects were further screened for their specific response to the 37 overlapping 18mer HpaA peptides at a final concentration of 5 μmol/L in an ICS assay. The identified 18mer sequences are shown, and the subsequently identified most potent sequences are in bold. A to E correspond to subjects 5750, 5250, 5751, 8901, and 5263 respectively. Gastroenterology 2013 144, 591-600DOI: (10.1053/j.gastro.2012.12.002) Copyright © 2013 AGA Institute Terms and Conditions

Figure 3 Detailed characterization of the immunodominant anti-HpaA CD4+ T-cell responses. (A) (i) The 13mer overlapping peptides within the HpaA190–207 18mers (subject 5750) were screened, with the 18mer results shown as open bars. (ii) Several N- and C-terminus extended and truncated peptides were titrated under serum-free conditions to compare their activities in the absence of serum proteases, and (iii–v) HLA class II antibodies and partial HLA class II matched BLCLs were used to identify the HLA allele presenting the HpaA192–204 13mer peptide. (B) (i) The 13mer peptides within HpaA82–105 (subject 5751) were screened as described in A. (ii) The most potent HpaA88–100 peptide and its amino acid truncated and extended variants were tested, and (iii–v) the HLA restriction profile for HpaA88–100 was determined using HLA class II antibodies and BLCLs. (C) (i) The 13mer peptides within the HpaA196–213 18mer (subject 5263) were screened as described in A. (ii–iv) HLA restriction of HpaA200–212 was analyzed by HLA class II antibody blocking and BLCLs. Gastroenterology 2013 144, 591-600DOI: (10.1053/j.gastro.2012.12.002) Copyright © 2013 AGA Institute Terms and Conditions

Figure 4 Ex vivo confirmation of the novel epitope-specific CD4+ T-cell responses. (A) CD4+ T cells isolated using a negative selection kit (Miltenyi Biotec) from whole PBMCs of subject 5751 were assessed for purity by flow cytometry. (B) HpaA154–171 or HpaA88–100 peptide-pulsed autologous mature DCs were used to stimulate purified CD4+ T cells in A for an overnight ELISPOT assay (ratio of DCs/CD4+ T cells, 1:10). (C) The naïve or memory CD4+ T cells purified by depleting CD45RO+ or CD45RA+ cells from the purified CD4+ T-cell population in A were assessed for purity by flow cytometry and (D) stimulated by HpaA88–100 in an overnight ELISPOT assay. Gastroenterology 2013 144, 591-600DOI: (10.1053/j.gastro.2012.12.002) Copyright © 2013 AGA Institute Terms and Conditions

Figure 5 Natural processing and presentation of the novel epitopes by APCs. (A) DCs (immature) and BLCLs were pulsed with recombinant HpaA and HP-WCL for 24 hours and then cocultured with HpaA192–204-specific T cells from subject 5750 for 5 hours in the presence of monensin. IFN-γ–secreting CD4+ T cells were determined by ICS. (B) HpaA88–100-specific T cells from subject 5751 were used to examine whether the epitope HpaA88–100 was a naturally processed peptide as described in A. Gastroenterology 2013 144, 591-600DOI: (10.1053/j.gastro.2012.12.002) Copyright © 2013 AGA Institute Terms and Conditions

Figure 6 Mapping immunodominant HpaA epitopes in PBMCs from HLA-DRB1*1501–positive subjects. HpaA-specific T-cell lines derived from 10 H pylori–infected subjects with HLA-DRB1*1501 expression were screened for their specific response to the 37 overlapping 18mer HpaA peptides and peptide HpaA88–100 at a final concentration of approximately 5 μmol/L in ICS assays. Gastroenterology 2013 144, 591-600DOI: (10.1053/j.gastro.2012.12.002) Copyright © 2013 AGA Institute Terms and Conditions

Figure 7 HpaA88–100-specific CD4+ T-cell response was associated with resistance to severe H pylori–associated gastric diseases. PBMCs from 59 H pylori–infected and HLA-DRB1*1501–expressing subjects with different gastric diseases were stimulated by peptide HpaA88–100 in vitro. HpaA88–100-specific CD4+ T-cell responses were assessed by ICS after 13 days of culture and the data analyzed according to gastric disease groups. Gastroenterology 2013 144, 591-600DOI: (10.1053/j.gastro.2012.12.002) Copyright © 2013 AGA Institute Terms and Conditions