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Amino-acid catabolizing enzymes produced by mononuclear phagocytes play a central role in regulating the immune response. The mammalian phenylalanine-catabolizing enzyme IL4-induced gene 1 (IL4I1) inhibits effector T lymphocyte proliferation and facilitates regulatory T-cell development. IL4I1 expression by macrophages of various human tumors may affect patient prognosis as it facilitates tumor escape from the T-cell response in murine models. Its enzymatic activity appears to participate in its effects, but some actions of IL4I1 remain unclear. Here, we show that the presence of IL4I1 during T-cell activation decreases early signaling events downstream of TCR stimulation, resulting in global T-cell inhibition which is more pronounced when there is CD28 costimulation. Surprisingly, the enzymatic activity of IL4I1 is not involved. Focal secretion of IL4I1 into the immune synaptic cleft and its binding to CD3 + lymphocytes could be important in IL4I1 immunosuppressive mechanism of action., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The Wilms tumor antigen, WT1, is associated with several human cancers, including leukemia. We evaluated WT1 as an immunotherapeutic target using our proven DNA fusion vaccine design, p.DOM-peptide, encoding a minimal tumor-derived major histocompatibility complex (MHC) class I-binding epitope downstream of a foreign sequence of tetanus toxin. Three p.DOM-peptide vaccines, each encoding a different WT1-derived, HLA-A2-restricted epitope, induced cytotoxic T lymphocytes (CTLs) in humanized transgenic mice expressing chimeric HLA-A2, without affecting hematopoietic stem cells. Mouse CTLs killed human leukemia cells in vitro, indicating peptide processing/presentation. Low numbers of T cells specific for these epitopes have been described in cancer patients. Expanded human T cells specific for each epitope were lytic in vitro. Focusing on human WT1(37-45)-specific cells, the most avid of the murine responses, we demonstrated lysis of primary leukemias, underscoring their clinical relevance. Finally, we showed that these human CTL kill target cells transfected with the relevant p.DOM-peptide DNA vaccine, confirming that WT1-derived epitopes are presented to T cells similarly by tumors and following DNA vaccination. Together, these data link mouse and human studies to suggest that rationally designed DNA vaccines encoding WT1-derived epitopes, particularly WT1(37-45), have the potential to induce/expand functional tumor-specific cytotoxic responses in cancer patients.

Interleukin-4-induced gene 1 (IL4I1) was first described as a B-cell IL4-inducible gene and is highly expressed in primary mediastinal B-cell lymphomas. We established stable HEK293 clones expressing human and mouse IL4I1 to examine their biochemical properties and function. Both proteins were secreted into the culture medium, and we observed the secretion of endogenous human IL4I1 (hIL4I1) protein in a mediastinal lymphoma B-cell line, MedB-1. We showed that IL4I1 has l-amino acid oxidase activity, optimal at physiological pH and primarily directed toward phenylalanine. Immunohistochemical analysis of secondary lymphoid organs showed staining of germinal center macrophages and inflammatory myeloid cells. In vitro, functional enzyme was highest in mature dendritic cells (DCs), suggesting a role in antigen-presenting cell/T-lymphocyte cross-talk. Indeed, hIL4I1 inhibited the proliferation of CD3-stimulated T lymphocytes with a similar effect on CD4(+) and CD8(+) T cells. In contrast, memory T cells were more strongly affected by hIL4I1 and its catabolite H(2)O(2) than naive T cells. hIL4I1 inhibitory effect was dependent on enzymatic activity and H(2)O(2) production and associated with a transient down-regulation of TCRzeta expression. Altogether these data suggest IL4I1 as a new immunomodulatory enzyme produced by DCs.

Lymphoma-derived immunoglobulin idiotype (Id) is a tumour-specific antigen used for antitumour vaccination in follicular lymphoma (FL). However, FL Ids are subject to hypermutation and subclones may escape antitumour cytotoxic T-cell response. To investigate the intraclonal epitope diversity, we sequenced the FL heavy chain gene (consensus Id gene) and subclones of 24 patients. The derived polypeptide sequences were analysed by bioinformatics for human leucocyte antigen (HLA)-A0201-restricted epitope prediction. Epitopes were classified according to BIMAS and SYFPEITHI scores. Surprisingly in these highly mutated polypeptides, the epitopes concentrated in short hotspots in the conserved framework regions (FRs), both in HLA-A0201(+) and HLA-A0201(-) patients. Similar hotspots have been observed by others in chronic lymphocytic leukaemia Ids which in contrast to FL have low mutation frequency. FR3 amino acids 78-88 displayed the best-score epitopes in Ids containing a VH3-family segment, the most represented in FL Ids. Such VH3-FR3(78-88) epitopes were previously demonstrated as immunogenic. Modifications of the epitope pattern in subclones of HLA-A0201(+) patients were generally absent from high-score peptides, including VH3-FR3(78-88) epitopes (83% unmodified). Therefore, no tendency for loss of HLA-A0201-restricted epitopes was evidenced and, given their limited intraclonal diversity, VH3-FR3(78-88) epitopes may provide a useful target for the induction of cytotoxic response in Id-vaccinated FL patients.