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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.

Trimethoprim-sulfamethoxazole (SXT)-resistant Staphylococcus aureus thymidine-dependent small-colony variants (TD-SCVs) are frequently isolated from the airways of cystic fibrosis (CF) patients, often in combination with isogenic normal strains if patients were treated with SXT for extended periods. As SXT inhibits the synthesis of tetrahydrofolic acid, which acts as a cofactor for thymidylate synthase (thyA), the survival of TD-SCVs depends exclusively on the availability of external thymidine. Since the underlying mechanism for thymidine dependency is unknown, we investigated if alterations in the thyA nucleotide sequences were responsible for this phenomenon. Sequence analysis of several clinical TD-SCVs and their isogenic normal strains with reference to previously published S. aureus thyA nucleotide sequences was performed. Three clinical TD-SCVs were complemented by transforming TD-SCVs with the vector pCX19 expressing ThyA from S. aureus 8325-4. Transcriptional analysis of metabolic and virulence genes and regulators (agr, hla, spa, citB, thyA, and nupC) was performed by quantitative reverse transcription-PCR. The previously published sequences of thyA and two normal clinical strains were highly conserved, while thyA of four normal strains and four SCVs had nonsynonymous point mutations. In 8/10 SCVs, deletions occurred, resulting in stop codons which were located in 4/10 SCVs close to or within the active site of the protein (dUMP binding). Complementation of TD-SCVs with thyA almost fully reversed the phenotype, growth characteristics, and transcription patterns. In conclusion, we demonstrated that mutations of the thyA gene were responsible for the phenotype of TD-SCVs. Complementation of TD-SCVs with thyA revealed that a functional ThyA protein is necessary and sufficient to change the SCV phenotype and behavior back to normal.