Hierarchy of alpha fetoprotein (AFP)-specific T cell responses in subjects with AFP-positive hepatocellular cancer

Abstract

We identified a series of immunodominant and subdominant epitopes from alpha 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*0201+AFP+ advanced stage hepatocellular cancer (HCC) patients, and have activated and expanded AFP-specific IFN-gamma-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-gamma-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.

FIGURE 1

Hierarchy of responses to AFP peptides in healthy donors. A, PBMC were cultured for 3 wk with autologous DC transduced with AdVhAFP and tested for peptide epitope recognition by IFN-γ ELISPOT. A representative experiment from one donor is shown. Responses to immunodominant epitopes AFP₁₃₇, AFP₁₅₈, AFP₃₂₅, and AFP₅₄₂ (left side) exceed those to subdominant epitopes (p < 0.001; two donors pooled). Response to PHA serves as a positive control; no spots were detected to K562/A2.1 cells (which presented peptides), and background spots to K562/A2.1 cells without peptide (“no peptide”) were low and were subtracted. B, PBMC were tested directly ex vivo, or after 7 days of IVS with AFP protein-fed DC or AdVhAFP-transduced DC in an IFN-γ ELISPOT for peptide epitope recognition. A representative experiment from one donor is shown. Responses to immunodominant epitopes (left side) exceed those to subdominant epitopes (p < 0.001, two donors pooled), and responses to AFP presented by AdVhAFP-transduced DC exceed those in response to AFP protein-fed DC. The same donor is shown in A and B to allow direct comparison between conditions.

FIGURE 2

Avidity of AFP peptide-specific CD8 T cells for peptide-pulsed targets. AFP peptide-specific T cells were expanded with 3 wk of culture with peptide-pulsed autologous DC. The T cells were plated in the IFN-γ ELISPOT with decreasing doses of peptide pulsed onto K562/A2.1 cells to determine the lowest amount of peptide required for IFN-γ production. Data are shown for the four immunodominant peptides and four subdominant peptides, as well as the positive control Flu M1 and negative control MART-1 peptide; cell cultures were from healthy donors.

FIGURE 3

Hierarchy of HCC patient responses to AFP peptides ex vivo. A, The hierarchy of response to AFP-derived peptides is shown for HCC patient A3. PBMC were thawed, and separated CD8 cells were plated directly in the IFN-γ ELISPOT with K562/A2.1 cells pulsed with individual peptides to determine the frequencies of peptide-specific T cells. T cells were detected to most of the peptides, with the highest frequency responses to AFP₁, AFP₂₁₈, and AFP₁₅₈. B, The hierarchy of response to AFP-derived peptides is shown for HCC patient B1. Cells were plated as above and the only responses were to epitopes AFP₁₃₇, AFP₁, AFP₃₀₆, and AFP₅₄₂. C, The median frequencies to each AFP peptide, for all six HCC patients (A3, A4, B1, B10, B11, B12) are shown; error bars indicate the first and fourth quartiles. The two-sided Wilcoxon test shows that, together, the six HCC patients have higher frequencies of T cells to the four immunodominant peptides, compared with the sum of the subdominant peptide frequencies (p = 0.023).

FIGURE 4

Hierarchy of HCC patient responses to AFP peptides after 7 days of IVS with AFP protein-fed DC. A, The hierarchy of response to AFP-derived peptides is shown for HCC patient A3. PBMC were cultured for 7 days, and separated CD8 cells were plated directly in the IFN-γ ELISPOT with K562/A2.1 cells pulsed with individual peptides to determine the frequencies of peptide-specific T cells. T cells were detected to many of the peptides, with the highest frequency responses to AFP₁₃₇, AFP₁₅₈, and AFP₂₃₅. B, The hierarchy of response to AFP-derived peptides is shown for HCC patient B1. Cells were plated as above, and the highest frequency responses were to subdominant epitopes AFP₂₁₈, AFP₃₀₆, AFP₄₈₅, and AFP₅₀₇. C, The median frequencies to each AFP peptide (after 7-day IVS) for all six HCC patients (A3, A4, B1, B10, B11, B12) are shown; error bars indicate the first and fourth quartiles. The two-sided Wilcoxon test shows that together, the six HCC patients have higher frequencies of T cells to the four immunodominant peptides, compared with the sum of the subdominant peptide frequencies, after 7-day IVS with protein-fed DC (p = 0.0072).

FIGURE 5

Hierarchy of HCC patient responses to AFP peptides after 7 days of IVS with AdVhAFP-transduced DC. A, The hierarchy of response to AFP-derived peptides is shown for HCC patient A3. PBMC were cultured for 7 days, and separated CD8 cells were plated directly in the IFN-γ ELISPOT with K562/A2.1 cells pulsed with individual peptides to determine the frequencies of peptide-specific T cells. T cells were detected to all of the peptides, with similar high-frequency responses. B, The hierarchy of response to AFP-derived peptides is shown for HCC patient B1. Cells were plated as above, and the highest frequency responses were to epitopes AFP₁₅₈, AFP₅₀₇, AFP₁, and AFP₄₈₅. C, The median frequencies to each AFP peptide (after 7-day IVS) for all six HCC patients (A3, A4, B1, B10, B11, B12) are shown; error bars indicate the first and fourth quartiles. The two-sided Wilcoxon test shows that, together, the six HCC patients do not have higher frequencies of T cells to the four immunodominant peptides after 7-day IVS with AdVhAFP-transduced DC (p = 0.56).

FIGURE 6

AFP T cell responses to the AFP-expressing, HLA-A2⁺ HCC cell line HepG2. A, Expanded AFP peptide-specific T cells (from healthy donor cultures shown in Fig. 2) were plated in the IFN-γ ELISPOT assays to determine the frequency of CD8 cells recognizing the HCC cell line HepG2, which endogenously produces AFP. Each AFP peptide-specific T cell culture recognized the tumor cells by producing IFN-γ. B, PBMC from each HCC patient were tested for recognition of HepG2 cells by ELISPOT. Data are shown for direct ex vivo analysis (from the experiments shown in Fig. 3), for 7-day IVS with protein-fed DC (“AFP/DC” (from the experiments shown in Fig. 4)), and for 7-day IVS with AdVhAFP-transduced DC (“AdVhAFP/DC” (from the experiments shown in Fig. 5)).