Common Ewing sarcoma-associated antigens fail to induce natural T cell responses in both patients and healthy individuals

Abstract

Disseminated or relapsed Ewing sarcoma (EwS) has remained fatal in the majority of patients. A promising approach to preventing relapse after conventional therapy is to establish tumor antigen-specific immune control. Efficient and specific T cell memory against the tumor depends on the expansion of rare T cells with native specificity against target antigens overexpressed by the tumor. Candidate antigens in EwS include six-transmembrane epithelial antigen of the prostate-1 (STEAP1), and the human cancer/testis antigens X-antigen family member 1 (XAGE1) and preferentially expressed antigen in melanoma (PRAME). Here, we screened normal donors and EwS patients for the presence of circulating T cells reactive with overlapping peptide libraries of these antigens by IFN-γ Elispot analysis. The majority of 22 healthy donors lacked detectable memory T cell responses against STEAP1, XAGE1 and PRAME. Moreover, ex vivo detection of T cells specific for these antigens in both blood and bone marrow were limited to a minority of EwS patients and required nonspecific T cell prestimulation. Cytotoxic T cells specific for the tumor-associated antigens were efficiently and reliably generated by in vitro priming using professional antigen-presenting cells and optimized cytokine stimulation; however, these T cells failed to interact with native antigen processed by target cells and with EwS cells expressing the antigen. We conclude that EwS-associated antigens fail to induce efficient T cell receptor (TCR)-mediated antitumor immune responses even under optimized conditions. Strategies based on TCR engineering could provide a more effective means to manipulating T cell immunity toward targeted elimination of tumor cells.

Fig. 1

STEAP1- and PRAME-reactive T cells are present in PB of healthy donors. IFN-γ Elispot analysis after stimulation of PBMCs with STEAP1, XAGE1 and PRAME PepMix. PBMCs were plated at a density of 4 × 10⁵ cells/well in the presence of 1 µg/ml of the respective PepMix or in medium alone. Actin PepMix stimulation was used as negative control, and VZV PepMix was used as positive control. Shown are the three tumor antigen-specific T cell responses against PRAME (left panel) or STEAP1 (middle and right panel) detected in 3 out of the 20 donors. Stars indicate positive responses by definition (see Methods)

Fig. 2

XAGE1- and PRAME-specific T cells detected in PB and BM of EwS patients following nonspecific prestimulation. Cryopreserved MCs from PB or BM were analyzed by IFN-γ Elispot either directly or on day 14 after nonspecific prestimulation with OKT-3 and anti-CD28 antibody for 48 h and subsequent expansion. Prestimulated cells were plated at 4 × 10⁵ cells/well in the presence of STEAP1, XAGE1 or PRAME PepMix (1 µg/ml), or in medium alone. Actin PepMix stimulation was used as a negative control, and nonspecific stimulation with PMA and ionomycin was used as positive control. a Positive IFN-γ responses of PBMCs from EwS patient #14 (see Table 2 for reference) to XAGE1 and PRAME. b Positive IFN-γ response of PBMCs from patient #16 to PRAME. c Positive IFN-γ response of MCs from BM of patient #15 to coculture with XAGE1 and with autologous EwS cells. Stars indicate positive responses by definition (see Methods). d Lack of specific IFN-γ responses to tumor-associated antigens and autologous EwS cells in BM from patient #26

Fig. 3

Quantification of T cells coexpressing PD-1. T cells coexpressing PD-1 were quantified by flow cytometry among CD4+ T cells (left panel) and CD8+ T cells (right panel) in PB of healthy donors (n = 9) and EwS patients (n = 9), and in BM of EwS patients (n = 12)

Fig. 4

Phenotypes and antigen responses of STEAP1- and XAGE1-specific T cells expanded from healthy donors. Shown are representative experiments of three. a Effector T cell phenotypes of CD8+ and CD4+ T cells expanded in response to STEAP1 presentation by S-DC versus F-DC flow cytometry. b Antigen-specific IFN-γ secretion of in vitro expanded STEAP1- and XAGE1-specific T cells was analyzed by Elispot after 2–4 weekly stimulations. 2 × 10⁵ T cells/well were restimulated with the respective PepMix (1 µg/ml) as indicated. Shown are representative examples for STEAP1 (left panel) and XAGE1 (right panel) of three each. Actin Pepmix stimulation was used as a negative control, and nonspecific stimulation with PMA and ionomycin was used as a positive control. c The cytolytic potential of in vitro expanded STEAP1- (left panel) and XAGE1-specific T cells (right panel) was assessed by quantifying CD107a expression of CD3+ T cells after 4-hour coincubation with tumor cell targets coexpressing STEAP1 and XAGE1 (VH-64, A-4573, K562/aAPC) and with PepMix-loaded autologous PBMCs at a 1:1 stimulator-to-responder ratio. Stimulation with K562 cells, and medium alone were used as negative controls