Hierarchy of <em>α</em> Fetoprotein (AFP)-Specific T Cell Responses in Subjects with AFP-Positive Hepatocellular Cancer<sup><sup><a href="#FN1" rid="FN1" class=" fn">1</a></sup></sup>
Abstract
We identified a series of immunodominant and subdominant epitopes from α fetoprotein (AFP), restricted by HLA-A*0201, which are recognized by the human T cell repertoire. The four immunodominant epitopes have been tested for immunogenicity in vivo, in HLA-A*0201AFP advanced stage hepatocellular cancer (HCC) patients, and have activated and expanded AFP-specific IFN-γ-producing T cells in these patients, despite high serum levels of this self Ag. Here, we have examined the frequency, function, and avidity of the T cells specific for subdominant epitopes from AFP. We find that T cells specific for several of these epitopes are of similar or higher avidity than those specific for immunodominant epitopes. We then tested the peripheral blood of subjects ex vivo with different levels of serum AFP for the hierarchy of response to epitopes from this Ag and find that HCC patients have detectable frequencies of circulating IFN-γ-producing AFP-specific CD8 T cells to both immunodominant and subdominant epitopes. We find the immunodominant and subdominant peptide-specific T cells to be differentially expanded with different modes of Ag presentation. Whereas spontaneous and AFP protein-stimulated responses show evidence for immunodominance, AdVhAFP-transduced dendritic cell-stimulated responses were broader and not skewed. Importantly, these data identify subdominant epitopes from AFP that can activate high-avidity T cells, and that can be detected and expanded in HCC subjects. These subdominant epitope-specific T cells can also recognize tumor cells and may be important therapeutically.
Hepatocellular carcinoma (HCC)4 is one of the main causes of cancer deaths with a global incidence of over 500,000 new cases per year. Once diagnosed, HCC has no effective systemic therapy and an overall 6% probability of 5-year survival. We are investigating the use of α fetoprotein (AFP) as a T cell-mediated tumor rejection Ag for HCC. We identified HLA-A*0201-restricted peptide epitopes that were immunogenic and naturally processed and presented, and then confirmed that three of them were present on the surface of HLAA*0201AFP HCC cells by mass spectrometry (1–4). Four of the peptides were designated as “immunodominant” based on the finding that they showed the strongest and most reproducible IFN-γ production and cytotoxic function in vitro in T cell cultures from healthy donor cells (with either adenovirus human AFP (AdVhAFP)-transduced DC stimulation or peptide-pulsed PBMC stimulation). The same four peptides were identified as immunodominant based on immunization of HLA-A*0201 transgenic mice, although the exact order of immunodominance was not identical (2). In the majority of healthy donor T cell cultures, AFP542 was immunodominant to AFP158, and these were followed by AFP325 and AFP137. In the HLA-A*0201 transgenic mice, the order was AFP158, AFP542, AFP325, and AFP137. Subsequent murine studies, in which different modes of immunization (plasmid DNA, peptides in adjuvant, AdV) were compared with IFN-γ ELISPOT readouts, indicated that, although AFP158 remained most immunodominant, the immunodominance order varied with immunization strategy for the other three peptides (3). This was not unexpected, because each method of immunization uses different AFP Ag presentation modes. In addition, the binding of each peptide to MHC class I has different stabilization and kinetic properties (2).
Based on the preclinical data, six AFPHLA-A*0201 subjects were treated with four immunodominant AFP-derived peptides emulsified in Montanide, and we found that after vaccination, the frequency of circulating AFP-specific T cells increased (by MHC tetramer assay) and the frequency of IFN-γ-producing AFP-specific T cells also increased (by ELISPOT assay) (5). However, responses to AFP158 and AFP542 no longer appeared immunodominant in this cohort of patients. We recently completed treatment of 10 AFPHLA-A*0201 HCC subjects with AFP peptide-pulsed autologous dendritic cells (DC). As in the pilot peptide/Montanide trial, we have seen increased frequencies of circulating AFP-specific T cells and of IFN-γ-producing AFP-specific T cells after vaccination (18). In this trial, none of the four immunizing peptides appears to be immunodominant to any other.
When we originally screened candidate AFP epitopes, we identified 10 subdominant epitopes in addition to the 4 immunodominant (Table I). The responses to these epitopes have not been studied further. As suggested by others (6, 7), responses to subdominant epitopes might be more clinically relevant due to reduced effects of central and peripheral tolerance on such T cells. The effects of fetal Ag expression and the presence of tumor-derived Ag presence in serum on hierarchy are unknown. Here, we have investigated the avidity of subdominant epitope-specific T cells for peptide-pulsed and tumor targets and the hierarchy of CD8 T cell responses to the previously identified immunodominant and subdominant epitopes in subjects with different levels of high serum AFP. We wanted to determine whether HCC subjects have altered hierarchy of responses to this self tumor associated Ag, and more importantly, to determine whether “subdominant” epitopes could be more potent immunogens in these subjects and be the focus of future immunotherapy vaccines.
Table I
Dominant and subdominant peptidesa
| Amino Acid Start | Length | Sequence | Anchors | Designation |
|---|---|---|---|---|
| 1 | 9 | MKWVESIFL | 1 | Subdominant |
| 137 | 9 | PLFQVPEPV | 2 | Immunodominant |
| 158 | 9 | FMNKFIYEI | 2 | Immunodominant |
| 178 | 9 | ILLWAARYD | 1 | Subdominant |
| 218 | 9 | LLNQHACAV | 2 | Subdominant |
| 235 | 9 | FQAITVTKL | 1 | Subdominant |
| 306 | 10 | TTLERGQCII | 1 | Subdominant |
| 325 | 10 | GLSPNLNRFL | 2 | Immunodominant |
| 485 | 9 | CIRHEMTPV | 2 | Subdominant |
| 492 | 9 | PVNPGVGQC | 1 | Subdominant |
| 507 | 10 | NRRPCFSSLV | 1 | Subdominant |
| 542 | 9 | GVALQTMKQ | 1 | Immunodominant |
| 547 | 10 | TMKQEFLINL | 2 | Subdominant |
| 555 | 9 | NLVKQKPQI | 2 | Subdominant |
| 549 | 9 | KQEFLINLV | 1 | Cryptic |
Footnotes
This work was supported by Scientist Development Grant from the American Heart Association (0330102N), the University of Pittsburgh Cancer Institute, and the Henry L. Hillman Foundation.
Abbreviations used in this paper: HCC, hepatocellular carcinoma; AFP, α fetoprotein; DC, dendritic cell; IVS, in vitro stimulation.
Disclosures
L. H. Butterfield is a coinventor on patents covering the AFP peptides used in this study. The University of California, Los Angeles, CA is also involved with these patents.
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