A human dendritic cell-based method to identify CD[4.sup.+] T-cell epitopes in potential protein allergens - Mini-Monograph

Environmental Health Perspectives, Feb, 2003 by Marcia Stickler, Jeanette Mucha, Scott Power, Fiona Harding

Data analysis of the pooled Bt peptide sets was based on replication of responses in neighboring wells rather than between two separate replicates for each peptide.

Data analysis. A response to a peptide was tabulated as positive if the SI was > 2.95. SI values for each donor were compiled for each peptide set, and the percentages of responders are reported. The average background response rate for each peptide set was calculated by averaging the percentage response for all of the peptides in the set. Major epitopes are defined as having a percentage response that is 3-fold or more than the background. Statistical significance was calculated using Poisson statistics for the number of responders to each peptide within the data set. The response to a peptide was considered significant if the number of donors responding to the peptide was different from the Poisson distribution defined by the data set with a p < 0.05.

HLA associations. HLA-DR and -DQ types were analyzed for associations with responses to defined epitope peptides. We used a chi-squared analysis with one degree of freedom. Where an allele was present in both the responder and nonresponder pools, a relative risk was calculated.

Results

Epitope map of Brazil nut 2S storage protein. The epitope mapping assay was performed on a donor set of 92 community blood bank donors using peptides describing the heavy and light chains of Ber e 1 (Figure 1A). The light chain of Ber e 1 was described by peptides 1-6. The heavy chain was described by peptides 7-27. The overall background response to the peptides in this set was 4.27%. This background level is higher than our overall epitope mapping average (average of ~3% over ~20 proteins tested; data not shown). We have observed that background responses are higher when testing peptide sets derived from proteins with known human exposure rates (data not shown). One epitope (peptide 4 corresponding to amino acids 10-24) was found in the light-chain sequence that met the criterion of 3-fold the background response to this peptide set, returning a total percent of responders equal to 18.48%. Three other prominent regions were found in the heavy-chain protein at peptide 12 [amino acids 16-30 (9.78%)], peptide 16 [amino acids 28-42 (7.61%)], and peptide 20 [amino acids 40-55 (11.96%)] that encompass a nested set of epitopes in the heavy-chain polypeptide. The percentage of responses to epitopes 16-30 and 40-55 are less than 3-fold (12.8%) but are more than 2-fold the background rate. The third epitope (28-42) does not reach 2-fold the background. Figure 1B shows the distribution of responses within the Ber e 1 data set. Peptide 4 had the most responses tabulated, a total of 17 out of 92 tested donors. There were 11 responses to peptide 20. This response rate to peptide 4 was highly significant, with p < 0.0001. The response to peptide 20 was also significant, with p = 0.02.

[FIGURE 1 OMITTED]

HLA associations within the Ber e 1 data set. The HLA-DR and -DQ types of donors who responded to peptides 4 (light-chain amino acids 10-24) and 20 (heavy-chain amino acids 40-55) were analyzed for the presence of any statistically significant enrichment in HLA subtypes. HLA-DQ2 was significantly associated with a nonresponse to peptide 4 (p = 0.05, relative risk = 0.29). There was a positive association between the presence of HLA-DR15(2) and a response to peptide 20 (p = 0.04, relative risk = 3.0). Interestingly, the strongest association found was a positive association between a response to peptide 12 and the presence of HLA-DR13 (p = 0.01, relative risk = 4.44).