Spontaneous and vaccine induced AFP-specific T cell phenotypes in subjects with AFP-positive hepatocellular cancer

Abstract

We are investigating the use of Alpha Fetoprotein (AFP) as a tumor rejection antigen for hepatocellular carcinoma (HCC). We recently completed vaccination of 10 AFP+/HLA-A2.1+ HCC subjects with AFP peptide-pulsed autologous dendritic cells (DC). There were increased frequencies of circulating AFP-specific T cells and of IFNgamma-producing AFP-specific T cells after vaccination. In order to better understand the lack of association between immune response and clinical response, we have examined additional aspects of the AFP immune response in patients. Here, we have characterized the cell surface phenotype of circulating AFP tetramer-positive CD8 T cells and assessed AFP-specific CD4 function. Before vaccination, HCC subjects had increased frequencies of circulating AFP-specific CD8 T cells with a range of naïve, effector, central and effector memory phenotypes. Several patients had up-regulated activation markers. A subset of patients was assessed for phenotypic changes pre- and post-vaccination, and evidence for complete differentiation to effector or memory phenotype was lacking. CD8 phenotypic and cytokine responses did not correlate with level of patient serum AFP antigen (between 74 and 463,040 ng/ml). Assessment of CD4+ T cell responses by ELISPOT and multi-cytokine assay did not identify any spontaneous CD4 T cell responses to this secreted protein. These data indicate that there is an expanded pool of partially differentiated AFP-specific CD8 T cells in many of these HCC subjects, but that these cells are largely non-functional, and that a detectable CD4 T cell response to this secreted oncofetal antigen is lacking.

Fig. 1

Diagram of the gating strategy used for multi-parameter flow cytometric analysis. Patient cells were thawed, treated with DNAse, and the adherent cells were removed. The CD8+ cells were purified and stained with MHC tetramers and additional antibodies. For analysis, lymphocytes are gated on by FSC/SSC, then CD5+ and CD8+ cells are selected. The additional markers were compared for % positivity and MFI (mean fluorescence intensity) for both total CD8+ and CD8+/tetramer+ cells. The example is for patient B10, whose AFP₁₃₇ specific T cells are shown for central (CCR7, CD62L) and peripheral (CCR5, CCR6) trafficking markers

Fig. 2

AFP tetramer positive T cell frequencies and phenotype. The percentage of tetramer positive cells detected pre-vaccination is shown. a Tetramer positive cells were stained for CD45RA and CCR7 and were divided into CD45RA+/CCR7+ (naïve), CD45RA+/CCR7- (effector memory), CD45RA-/CCR7+ (central memory), and CD45RA-/CCR7- (effector) groups. b CD45RA was compared with CD27 and CD28 to further examine differentiation. The representative HCC patients A4, B3 and B10 are shown, as well as healthy donor controls

Fig. 3

AFP tetramer-positive T cell activation phenotype pre-vaccination. The expression of HLA-DR, CD69, CD25 on AFP_137–145, AFP_158–166, and AFP_325–334 tetramer positive cells is shown. The representative HCC patients A4, B3 and B10 are shown, as well as healthy donor controls

Fig. 4

AFP tetramer-positive T cell trafficking phenotype pre-vaccination. The expression of central trafficking markers CCR7 and CD62L and peripheral trafficking markers CCR5 and CCR6 on AFP_137–145, AFP_158–166, and AFP_325–334 tetramer positive cells is shown. The representative HCC patients A4, B3 and B10 are shown, as well as healthy donor controls

Fig. 5

HCC patient changes in activation markers with vaccination. Patients treated with AFP peptide-pulsed autologous DC were examined before, during (B11, B12) and after vaccination (all 4 patients) for alterations of activation markers HLA-DR, CD69, CD25. Markers on AFP_137–145, AFP_158–166, and AFP_325–334 tetramer positive cells are shown. Increases >10% of baseline value are marked with an asterisk

Fig. 6

AFP-specific CD4 multi-cytokine ELISPOT analysis. ELISPOT assays were performed to detect IFNγ, IL-2, IL-5 and IL-10 from CD4 purified T cells stimulated with autologous DC pulsed for 1 h with AFP protein (AFP + DC). These were compared to unpulsed DC (0 + DC) and PHA positive control. Cytokines produced by DC plated alone (DC only) are also shown