Natural immunity to pluripotency antigen OCT4 in humans

Abstract

OCT4 is a transcription factor critical for the pluripotency of human embryonal stem (ES) and induced pluipotency stem (IPS) cells. OCT4 is commonly expressed in germ-cell tumors as well as putative cancer stem cells in several tumors, and is a key determinant of oncogenic fate in germ-cell tumors. The capacity of the human immune system to recognize this critical stem-cell gene is not known, but has implications for preventing tumors with ES/IPS-based therapies and targeting stem-cell pathways in cancer. Here we show that OCT4-specific T cells can be readily detected in freshly isolated T cells from most (>80%) healthy donors. The reactivity to OCT4-derived peptides resides primarily in the CD45RO(+) memory T-cell compartment and consists predominantly of CD4(+) T cells. T cells reactive against OCT4-derived peptides can be readily expanded in culture using peptide-loaded dendritic cells. In contrast to healthy donors, immunity to OCT4 was detected in only 35% of patients with newly diagnosed germ-cell tumors. However, chemotherapy of germ-cell tumors led to the induction of anti-OCT4 immunity in vivo in patients lacking such responses at baseline. These data demonstrate the surprising lack of immune tolerance to this critical pluripotency antigen in humans. Harnessing natural immunity to this antigen may allow immune-based targeting of pluripotency-related pathways for prevention of cancers, including those in the setting of ES/IPS-based therapies.

Fig. 1.

Immunity to OCT4 in freshly isolated PBMCs from healthy blood donors. (A and B) Reactivity of freshly isolated human PBMCs from healthy donors to OCT4 peptide library: 2 × 10⁵ PBMCs were cultured alone or in the presence of 3 μg/mL of OCT4 peptide mixes, or PHA, as a control. After 48 h, the culture supernatant was harvested and analyzed for the presence of IP10 by Luminex assay. (A) Data for representative healthy donors with or without detectable reactivity to OCT4 peptide library. (B) Data for reactivity to the positive OCT4 mix (represented as fold-change in reactive mix compared with control) in all 30 donors tested. Based on interassay and intra-assay variance, ≥2-fold increase in IP10 production (in reactive mix versus control), with a minimal absolute measurement of 100 pg/mL, was predetermined to be positive for the presence of antigen-specific T cells, as discussed under Materials and Methods. (C) Bulk PBMCs or those depleted of CD3⁺ T cells were cultured alone (control) or with OCT4 peptide library. After 48 h, the culture supernatant was harvested and analyzed for the presence of IP10 by Luminex assay. Data are representative of similar experiments on three donors. (D) PBMCs were cultured alone or OCT4 peptide mix in the presence of either anti-IFN-γ blocking antibody or isotype control antibody. After 48 h, the culture supernatant was harvested and analyzed for the presence of IP10 by Luminex assay. Data are representative of similar experiments on three donors.

Fig. 2.

Antigen dependent proliferation of OCT4-specific memory T cells. (A) Proliferative response to OCT4 peptide library: 2 × 10⁵ PBMCs were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and cultured with 1 μg/mL of anti-CD28 and anti-CD49d antibody alone (control) or in the presence of OCT4-derived peptides (3 μg/mL). (Left) Proliferation of CD3⁺-gated cells in a representative donor. Note the predominant proliferation of CD4⁺ T cells. (Right) Antigen-dependent proliferation in six donors with or without reactivity in the IP10 assay, as in Fig 1A. (B) PBMCs were depleted of CD45RO⁺ T cells using immunomagnetic beads (or left undepleted) before stimulation with OCT4 peptides (Left), or PHA as a control (Right).

Fig. 3.

Fine specificity of OCT4-specific T-cell immunity and activation of peptide-specific T cells. (A) Reactivity to individual peptides: PBMCs were cultured with individual peptides from submix 3 to determine the specific reactive peptide. After 48 h, culture supernatant was harvested and analyzed for the presence of IP10 by Luminex assay. The figure shows the reactivity against individual peptides for 10 healthy donors. (B) Expansion of peptide-reactive T cells with peptide-pulsed DCs. Monocyte-derived mature DCs loaded with active peptide (as identified in experiments in Fig 2A) were used to stimulate autologous T cells. The presence of peptide-specific IFNγ-producing T cells was analyzed by intracellular cytokine flow cytometry following restimulation with anti-CD28 and anti-CD49d, with or without stimulating peptide. Data shown are representative of four different donors.

Fig. 4.

Immunity to OCT4 in patients with GCTs. (A) Reactivity of freshly isolated human PBMCs from newly diagnosed GCT patients (n = 21) or healthy controls (n = 30) to OCT4 peptide library (as in Fig 1B) or PHA as a control. Positive reactivity was predetermined to be ≥2-fold increase in IP10 production (in reactive mix versus control), with a minimal absolute measurement of 100 pg/mL, as discussed under Materials and Methods. (B) Effect of antitumor therapy on OCT4 immunity: PBMCs from GCT patients (n = 12) were analyzed at baseline and at the completion of therapy for reactivity to OCT4 and to viral antigens (CEF) as a control. Data shown are reactivity at baseline and at the completion of therapy. Bold lines represent mean reactivity.