Giuseppe Consogno

Increased interleukin-10 serum levels in patients with solid tumours

In 40 out of 99 patients (40.4%) with solid tumours of different tissue, but the same stage (IV), elevated serum levels of interleukin-10 were observed. The mean levels of the cytokine in patients with malignant melanoma (24.3 ng/ml), pancreatic (6.8 ng/ml) or gastric (6.3 ng/ml) adenocarcinoma were significantly higher than in healthy subjects (3.4 ng/ml) or in patients with uterine fibroma (1.7 ng/ml). Patients with colon (6.8 ng/ml) and renal (5.7 ng/ml) carcinoma had similar values of interleukin-10 but did not significantly differ from controls. Interleukin-10 is known to suppress the functions of both T lymphocytes and macrophages, working as a general dampener of the immune and inflammatory responses. The observation of increased circulating levels of interleukin-10 in cancer patients may have important implications for future investigations, immunological monitoring and therapeutic intervention on neoplastic patients, and suggests a mechanism for tumour cells escaping from immune surveillance.

Identification of Novel Subdominant Epitopes on the Carcinoembryonic Antigen Recognized by CD4+ T Cells of Lung Cancer Patients

The carcinoembryonic Ag (CEA) is an attractive target for immunotherapy because of its expression profile and role in tumor progression. To verify the existence of spontaneous anti-CEA CD4+ T cells in lung cancer patients, we first identified CEA sequences forming naturally processed epitopes, and then used the identified epitopes to test their recognition by CD4+ T cells from the patients. We had previously identified CEA177–189/355–367 as an immunodominant epitope recognized by CD4+ T cells in association with several HLA-DR alleles. In this study, we identified four additional subdominant CEA sequences (CEA99–111, CEA425–437, CEA568–582, and CEA666–678), recognized in association with one or more HLA-DR alleles. Peptide-specific CD4+ T cells produced proinflammatory cytokines when challenged with the native protein and CEA-expressing tumor cells, thus demonstrating that the identified CEA sequences contain naturally processed epitopes. However, CEA is expressed in the thymus and belongs to the CD66 family that comprises highly homologous molecules expressed on hemopoietic cells, raising concerns about tolerance interfering with the in vivo development of anti-CEA immunity. We thus tested the spontaneous reactivity to the identified epitopes of peripheral blood CD4+ T lymphocytes from eight early-stage lung cancer patients bearing CEA-positive tumors. We found GM-CSF- and IFN-γ- producing CD4+ T cells in two patients. Our data indicate that CD4+ immune responses against CEA develop in neoplastic patients, suggesting that tolerance toward CEA or cross-reactive CD66 homologous molecules might be either not absolute or be overcome in the neoplastic disease.

Cytokine secretion associated with the clearance of apoptotic bodies in renal cell carcinoma patients

The factors determining the outcome of immunotherapy in metastatic renal cell carcinoma (RCC) patients remain elusive. Macrophages from normal donors that phagocytose apoptotic cells secrete the immunosuppressive cytokine IL‐10 in vitro. Conversely, IL‐10 genetic deletion enhances the immunogenicity of apoptotic tumor cells in vivo. Elevated pre‐treatment levels of IL‐10 are associated with an unfavorable outcome of RCC. We examined whether the ability to release IL‐10 by macrophages from RCC patients that phagocytosed apoptotic cells correlated with the outcome of immunotherapy. To this aim, we derived macrophages from 30 patients with metastatic RCC and from 21 healthy subjects (11 sex‐ and age‐matched healthy controls and 10 younger donors). Patients either had a clinical response after immunotherapy, with a median survival after treatment of more than 18 months (n = 16), or were beginning immunotherapy after diagnosis of metastatic disease (n = 14). Macrophages from responding patients challenged with apoptotic cells released significantly less IL‐10 than controls (p = 0.0075) and recently diagnosed patients (p = 0.0198), as ascertained by a 2‐sided Students t‐test. This was not because macrophages from responding patients lost the ability to secrete IL‐10, because antibody opsonization of apoptotic cells rescued IL‐10 secretion. In contrast, macrophages from all groups of donors released similar amounts of TNF‐α. The failure in IL‐10 secretion by engulfing macrophages of responding subjects may exalt the immunogenicity of dying tumor cells, contributing to the success of immunotherapy.

Role of Interleukin-2 in Regulating Lymphocyte Activation and Recirculation

TO UNDERSTAND better the immunological reactions primed by interleukin-2 (IL-2) in cancer patients we have investigated the biological effects of systemic rIL-2 on the circulating lymphocytes of cancer patients [ 1, 21. We report here immunological tests, either functional or phenotypic, in 8 patients with advanced renal cell carcinoma treated with rIL-2 (18 x lo6 U/m*/day) by continuous intravenous administration for 5 consecutive days. Heparinised blood samples were drawn immediately before starting the first treatment cycle, 24 and 96 h after starting the rIL-2 infusion, and 2 days after the end of the infusion (168 h). Mononuclear cells were obtained by centrifugation over Ficoll gradient and resuspended in RPM1 1640 medium (Biochrom, Berlin, FRG). Phenotypic analysis was performed in a direct, doubie-staining, immunofluorescence assay. During lymphopenia, induced by the IL-2 administration, we observed a progressive decrease in the percentage of theCD3+CD4+ T-lymphocytes, while theCD3+CD8+ subset did not change significantly. Small CD4+ and CDS+ T cells are extracted by high endothelial venules with different efficiency (higher for the CD4+ than for the CD8+ subset) {3] and the rIL-2 infusion seems to emphasise this difference. The hypothesis that the decrease of the CD4+ T cells might be due to increased extravasation, is also sustained by concomitant reduced proliferation of the patients’ peripheral blood mononuclear cells (PBMC) cultured for 3 days with different concentrations of phytohaemaglutinin (PI-IA) or concavalin A (ConA) (Sigma Chemical Co, St. Louis, Missouri). On stopping the rIL-2 infusion, when a rebound lymphocytosis is observed, the percentage of the CD4+ T-lymphocytes, and the capacity of