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. 2007 Mar 28:5:17.
doi: 10.1186/1479-5876-5-17.

CMV pp65 and IE-1 T cell epitopes recognized by healthy subjects

Stefanie L Slezak  1 , Maria Bettinotti, Silvia Selleri, Sharon Adams, Francesco M Marincola, David F Stroncek
Affiliations

CMV pp65 and IE-1 T cell epitopes recognized by healthy subjects

Stefanie L Slezak et al. J Transl Med. .

Abstract

Background: Adoptive immune and vaccine therapies have been used to prevent cytomegalovirus (CMV) disease in recipients of hematopoietic progenitor cell transplants, but the nature of T cell responses to CMV have not been completely characterized.

Methods: Peptide pools and individual peptides derived from the immune-dominant CMV proteins pp65 and IE-1 and antigen-specific, cytokine flow cytometry were used to characterize the prevalence and frequency of CD4+ and CD8+ memory T cells in 20 healthy CMV-seropositive subjects.

Results: CD8+ T cell responses to pp65 were detected in 35% of subjects and to IE-1 in 40% of subjects. CD4+ T cell responses to pp65 were detected in 50% of subjects, but none were detected to IE-1. Several new IE-1 HLA class I epitopes were identified, including 4 restricted to HLA-C antigens. One region of IE-1 spanning amino acids 300 to 327 was rich in class I epitopes. The HLA class I restrictions of IE-1 peptides were more promiscuous than those of pp65 peptides.

Conclusion: Since naturally occurring CD4+ and CD8+ T cell responses to pp65 were detectable in many subjects, but only CD8+ T cell responses to IE-1 were detected, pp65 may be better than IE-1 for use in vaccine and adoptive immune therapies.

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Figures

Figure 1
Figure 1
Identification of CMV pp65 epitopes using 15-mer and nanomer peptides. PBMCs were stimulated with a library of 138 15-mer peptides overlapping by 11 residues and covering the entire pp65 protein. IFN-γ flow cytometry was used to detect T cell responses. To identify specific epitopes, PBMCs were stimulated with subpools containing 10 overlapping 15-mer peptides. PBMCs from donors reactive with CD4+ T cells were tested further with individual 15-mers. The results of analysis of CD4+ T cell responses in donor 14 to pp65 peptides are shown.
Figure 2
Figure 2
Identification of CMV IE-1 epitopes using 15-mer and nanomer peptides. PBMCs were stimulated with a library of 120 15-mer peptides overlapping by 11 residues and covering the entire IE-1 protein. IFN-γ flow cytometry was used to detect T cell responses. To identify specific epitopes, PBMCs were stimulated with peptide subpools containing 10 overlapping 15-mer peptides. PBMCs from donors with reactive CD8+ T cells were tested further with individual 15-mers and nanomers. The results of analysis of CD8+ T cell responses in donor 6 to IE-1 peptides are shown.
Figure 3
Figure 3
Identification of CMV pp65 15-mer and nanomer epitopes recognized by CD8+ T cells. PBMCs from CMV-seropositive subjects reactive with peptides in a pp65 subpool were tested against all the individual 15-mers in each subpool. Following the identification of reactive 15-mers, nanomers overlapping at 8 amino acids and spanning the reactive 15-mer peptides were tested against PBMCs from the reactive subject. The reactive 15-mer and nanomer peptides are shown. Testing of cells from 7 subjects led to the identification of 6 pp65 15-mers reactive with CD8+ T cells. Testing of the overlapping nanomers identified 5 epitopes. Cells from donor 3 were not available to test with the nanomer peptides. Although CD8+ T cells from donor 10 were reactive with peptides in subpool 6 and a reactive 15-mer, pp65221–235, was identified, no reactive nanomer was identified. NT = not tested.
Figure 4
Figure 4
Identification of CMV IE-1 15-mer and nanomer epitopes from 3 peptide subpools reactive with CD8+ T cells. PBMCs from 4 donors were reactive with peptides in IE-1 subpools 3, 5, and 10. Testing of all 15-mers in each subpool identified 3 nanomers reactive with CD8+ T cells, one from each subpool. Testing of the 6 overlapping nanomers spanning each reactive 15-mer lead to the identification of 3 nanomer epitopes.
Figure 5
Figure 5
Identification of CMV pp65 15-mer and nanomer epitopes from peptide subpool 8 that were reactive with CD8+ T cells. Testing of IE-1 15-mers from subpool 8 against cells from donor 2 (gold bar), donor 5 (red bar), donor 6 (light blue bar), and donor 7 (navy blue bar) revealed that the peptide IE-1297–311 was reactive with donor 6, IE-1305–319 was reactive with donor 7, IE-1309–323 was reactive with donors 2 and 6, and IE-1313–327 was reactive with donor 5 (Panel A). Testing of the 6 nanomers overlapping IE-1297–311 against cells from donor 6 revealed that IE-1300–308 was the dominant nanomer (Panel B). Testing of the nanomers spanning the overlapping 15-mers IE-1305–319, IE-1309–323, and IE-1313–327 against PBMCs from donors 2, 5, and 7 identified 4 nanomers that were reactive with CD8+ T cells: IE-1305–313, IE-1308–316, IE-1312–320 and IE-1319–327, (Panel C).
Figure 6
Figure 6
Identification of CMV pp65 15-mer epitopes from 3 peptide subpools that were reactive with CD4+ T cells. PBMCs from 8 donors were reactive with peptides in subpools 6, 8, and 10. Testing of all 15-mers in each subpool identified three15-mers that were reactive with CD4+ T cells, one from each subpool.
Figure 7
Figure 7
Identification of CMV pp65 15-mer epitopes from peptide subpool 13 that were reactive with CD4+ T cells. PBMCs from 5 donors were reactive with pp65 peptides in subpool 13. Testing of all 15-mers in subpool 13 identified three 15-mers that were reactive with CD4+ T cells, pp65489–503, pp65505–519, and pp65509–523.
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