Alterations of the immunosuppressive IL4I1 enzyme activity induced by naturally occurring SNP/mutations.

The immunosuppressive phenylalanine oxidase interleukin 4-induced gene 1 (IL4I1), primarily produced by antigen-presenting cells, inhibits T-cell proliferation and promotes the generation of Foxp3(+) regulatory T cells in vitro. Highly expressed by tumour-associated macrophages from human cancers, IL4I1 has a potential role in immune evasion from the anti-tumour immune response. We have reviewed single-nucleotide polymorphisms (SNPs) and mutations described for the exon 4 of the IL4I1 isoform 1, which is expressed in lymphoid tissue. Two of them were expressed in an exogenous system to analyse their effect on the enzymatic activity. The N92D SNP leads to a hyperactive enzyme, while the R102G mutation is hypomorphic. Moreover, we show that IL4I1 activity is not only directed against phenylalanine, as initially described, but also at a lower level against arginine. These data pave the way to more extensive analyses of the mutational state of IL4I1 in pathological conditions such as cancer, where its participation in immune system dysfunctions may have therapeutic implications.

Two atypical cases of cutaneous gamma/delta T-cell lymphomas.

Cutaneous γ/δ T-cell lymphoma (CGD-TCL) is a recent entity described in the newly revised World Health Organization-European Organization for Research and Treatment of Cancer classification of cutaneous lymphomas, and is characterized by the γ/δ T-cell receptor expression on atypical lymphocytes. Only a few cases of primary CGD-TCL have been reported, with an extremely aggressive course (median survival time of 15 months). We describe 2 atypical cases of CGD-TCL. The first case was initially misdiagnosed as an inflammatory panniculitis due to the granulomatous infiltrate on the skin biopsy specimen. Diagnosis was confirmed using δ PCR that revealed γ/δ T-cell clonal expansion. The evolution was marked by predominant γ/δ T-cell infiltrate with diffuse body fat involvement as seen on positron emission tomography-computed tomography. The second case is the first described Epstein-Barr virus (EBV)-associated CGD-TCL with a rapidly fatal evolution. CGD-TCL is also a heterogeneous entity and δ PCR and EBV-encoded RNA probe to detect an EBV latent infection may help diagnose and characterize these cutaneous lymphomas.

Phase I trial of recombinant adenovirus gene transfer in lung cancer. Longitudinal study of the immune responses to transgene and viral products.

Animal studies indicate that the use of replication-deficient adenovirus for human gene therapy is limited by host antivector immune responses that result in transient recombinant protein expression and blocking of gene transfer when rechallenged. Therefore, we have examined immune responses to an adenoviral vector and to the beta-galactosidase protein in four patients with lung cancer given a single intratumor injection of 10(9) plaque-forming units of recombinant adenovirus. The beta-galactosidase protein was expressed in day-8 tumor biopsies from all patients at variable levels. Recombinant virus DNA was detected by PCR in day-30 and day-60 tumor biopsies from all patients except patient 1. A high level of neutralizing antiadenovirus antibodies was detected in patient 1 before Ad-beta-gal injection whereas it was low (patient 3) or undetectable in the other two patients. All patients developed potent CD4 type 1 helper T cell (Th1) responses to adenoviral particles which increased gradually over time after injection. Antiadenovirus cytotoxic T lymphocyte responses were consistently boosted in the two patients examined (patients 3 and 4). Sustained production of anti-beta-galactosidase IgG was observed in all patients except patient 1. Consistent with anti-beta-gal antibody production, all patients except patient 1 developed intense, dose-dependent Th1 responses to soluble beta-galactosidase which increased over time. Strong beta-galactosidase-specific cytotoxic T lymphocyte responses were detected in patients 2, 3, and 4. Our results clearly show that despite the intensity of antiadenovirus responses, transgene protein expression was sufficient to induce strong and prolonged immunity in three patients. Recombinant adenovirus injected directly into the tumor is a highly efficient vector for immunizing patients against the transgene protein.

Longitudinal follow-up of cellular and humoral immunity induced by recombinant adenovirus-mediated gene therapy in cancer patients.

Replication-defective adenoviruses are arousing growing interest as both gene therapy and vaccine vectors. In a phase I clinical trial designed to evaluate the feasibility and tolerance of recombinant adenovirus (rAd)mediated gene transfer, we previously demonstrated that a single intratumoral injection of 10(9) PFU of rAd encoding the beta-galactosidase protein (Ad-beta-Gal) induced strong short-term (1-3 months) humoral, helper (Th1 type) and cytotoxic T cell responses specific for the transgene product in patients with advanced lung cancer. The purpose of the present study was to evaluate the persistence of long-lasting immunity to the transgene protein and in parallel, to assess patient immunocompetence revealed by responses to recall antigens (tetanus toxoid, purified protein derivative), viral pathogens (Epstein-Barr virus, influenza virus), and allogeneic antigens in mixed lymphocytic reactions. The beta-Gal-specific proliferative response declined rapidly in patients with progressive disease, as did responses to the other antigens. In contrast, a long-lasting proliferative response to beta-gal was maintained in an immunocompetent patient in complete remission 2 years after an injection of 108 PFU of Ad-beta-Gal. Anti-beta-Gal humoral (IgG and IgA) responses persisted notably, as did responses to TT and poliomyelytic antigens. While T cell effector cytotoxic responses specific for the viral peptides plummeted, the frequency of anti-beta-Gal CTL precursors remained particularly high, thus attesting to major immunization. Despite the impact of both advanced disease and chemotherapy on immunocompetence, we show the long-term persistence of immunity to the transgene protein vectorized by rAd.

The novel immunosuppressive enzyme IL4I1 is expressed by neoplastic cells of several B-cell lymphomas and by tumor-associated macrophages.

We previously reported a strong IL4I1 gene expression in primary mediastinal B-cell lymphoma (PMBL) and recently identified the protein as a secreted L-phenylalanine oxidase, physiologically expressed by myeloid cells, which inhibits T-cell proliferation in vitro. Here, we analyzed the pattern of IL4I1 protein expression in 315 human lymphoid and non-lymphoid malignancies. Besides PMBL, IL4I1 expression in tumors was very frequent. IL4I1 was detected in tumor-associated macrophages from most of the tumors and in neoplastic cells from follicular lymphoma, classic and nodular lymphocyte predominant Hodgkin lymphomas and small lymphocytic lymphoma, three of which are germinal center derived. IL4I1-positive tumor cells were also detected in rare cases of solid cancers, mainly mesothelioma. The enzymatic activity paralleled protein expression, suggesting that IL4I1 is functional in vivo. Depending on the tumor type, IL4I1 may impact on different infiltrating lymphocyte populations with consequences on tumor evolution. In the particular case of follicular lymphoma cells, which are susceptible to antitumor cytotoxic T cells killing but depend on interactions with local T helper cells for survival, a high level of IL4I1 expression seems associated with the absence of bone marrow involvement and a better outcome. These findings plead for an evaluation of IL4I1 as a prognosis factor.

Immune response to recombinant adenovirus in humans: capsid components from viral input are targets for vector-specific cytotoxic T lymphocytes.

We previously demonstrated that a single injection of 10(9) PFU of recombinant adenovirus into patients induces strong vector-specific immune responses (H. Gahéry-Ségard, V. Molinier-Frenkel, C. Le Boulaire, P. Saulnier, P. Opolon, R. Lengagne, E. Gautier, A. Le Cesne, L. Zitvogel, A. Venet, C. Schatz, M. Courtney, T. Le Chevalier, T. Tursz, J.-G. Guillet, and F. Farace, J. Clin. Investig. 100:2218-2226, 1997). In the present study we analyzed the mechanism of vector recognition by cytotoxic T lymphocytes (CTL). CD8(+) CTL lines were derived from two patients and maintained in long-term cultures. Target cell infections with E1-deleted and E1-plus E2-deleted adenoviruses, as well as transcription-blocking experiments with actinomycin D, revealed that host T-cell recognition did not require viral gene transcription. Target cells treated with brefeldin A were not lysed, indicating that viral input protein-derived peptides are associated with HLA class I molecules. Using recombinant capsid component-loaded targets, we observed that the three major proteins could be recognized. These results raise the question of the use of multideleted adenoviruses for gene therapy in the quest to diminish antivector CTL responses.