Effects of Interferon-Gamma 1-b (IFN-γ) on Neutrophil Function and Biochemistry in Patients with Chronic Granulomatous Disease

Introduction: Chronic Granulomatous Disease (CGD) is a genetic disorder of the Nox2 enzyme system in phagocytic cells. Neutrophils and monocytes from patients with CGD are deficient in Nox2 components, unable to produce superoxide anion and other reactive oxygen species (ROS), and cannot kill certain types of bacteria and fungi. Patients with CGD suffer from recurrent, life-threatening infections. Treatment includes prophylaxis with antibiotic and antifungal agents and therapy with IFN-γ. Results from clinical trials of IFN-γ in patients with CGD have demonstrated decreased incidence and severity of infections. The exact mechanism(s) by which IFN-γ achieves its clinical effects is not clear. Previous studies from our laboratory in healthy adult volunteers demonstrated that administration of IFN-γ induced dramatic changes in the neutrophil phenotype with significant functional implications. To investigate IFN-γ induced changes in neutrophils further we initiated a study in CGD patients. Clinical Trial and Methods: Patients with CGD were enrolled on a study protocol approved by the COMIRB at the University of Colorado Denver. Patients between 5 and 60 years with defined genetic variants of CGD, but without recent infections or medical complications were entered onto the study. IFN-γ was stopped for a week to allow a drug washout period; prophylactic antibiotics and antifungal medications were continued. Blood samples were obtained from patients off IFN-γ, 12 hours after the first dose, and after the fourth dose of IFN-γ (50 mcg./m2 given on a standard schedule three times a week). Neutrophils were isolated by Dextran sedimentation, Ficoll Hypaque density centrifugation, and hypotonic lysis of red blood cells. RNA was isolated by standard technique and gene expression completed by RNAseq analysis (results pending). Ingestion was measured with fluorescently labeled S. aureus, bactericidal activity against S. aureus with 1:1 ratio of neutrophils to bacteria in 10% normal human serum by standard technique, and chemotaxis to fMLF and C5a with calcein-AM labeled neutrophils across fluorescence blocking PET membrane in Corning HTS Fluroblock plates. Superoxide anion was measured as SOD inhibitable cytochrome c reduction in response to standard agonists, CD11b expression and F-actin assembly in response to PMA and fMLF by flow cytometry, MHC Class II antigens by flow cytometry, nitric oxide (NO) levels in neutrophil lystes using a colorimetric assay to measure the combined nitrate and nitrite levels, and apoptosis determined as the combined caspase 3 and caspase 7 activity in isolated lysates using the Caspase-Glo 3/7 Assay from Promega. Results: We report here preliminary results on the first three patients studied; all are gp91phox deficient X-linked CGD. As expected, superoxide anion was not detected from patient neutrophils off INF-γ or during treatment. Ingestion and cell motility were not affected by IFN-γ administration. In all three subjects, caspase activity was decreased during IFN-γ treatment compared to before. In all three patients bactericidal activity against S. aureus was deficient; in 2/3 patients, administration of IFN was associated with a small but specific increase in killing, without change in ROS production. Strikingly, in two patients who showed improved bactericidal activity, NO production in lysates was increased after IFN-γ treatment compared to before and showed a further increase when cells were stimulated with fMLF. CD11b expression and F-actin assembly did not appear to be altered by IFN-γ treatment. Expression of HLA Class II markers (HLADRA and CD 274) appeared on neutrophils and increased in expression after initiation of IFN treatment. This change also was seen with CD 40. Summary: IFN-γ appeared to have dramatic effects on neutrophils from patients with CGD including possible improved bactericidal activity, the generation of NO which could be bactericidal and compensate for the deficiency of generation of ROS, and expression of MHC Class II antigens with possible association to antigen processing and enhanced interaction with adaptive immune function. Conclusion: Changes in the neutrophil phenotype induced by IFN-γ may provide alternative strategies compensating for the genetic deficiency of CGD. Defining how IFN-γ enhances neutrophil function may expand the use of this drug to other medical disorders in which innate immune response is involved. Disclosures Ambruso: Horizon Pharma Ireland Ltd.: Consultancy.

Immune evasion in colo-rectal cancer in a cohort of Sudanese patients: possible roles for MHC Class II antigens

Background: Colorectal cancer (CRC) is the third most common cancer world-wide. The majority of cases occur in the developed world. This prospective study aimed to correlate different human leukocyte antigens (HLA types; HLA DRB1 and DRB3) with the aggressiveness of CRC in Sudanese patients.Methods: Thirty-three patients with histopathologically confirmed CRC were included in the study. Demographic, clinical and laboratory data were recorded. Molecular typing for HLA DRB1 (DR1, 7 and 17), DRB3 and DRB4 were carried out using PCR-based Sequence-Specific Primers.Results: Forty percent of the patients were ≤ 50years; with a male to female ratio of 2.5:1. Rectal bleeding was the commonest presenting symptom. While moderately differentiated adenocarcinoma was the dominant histological type. Duke's stages B and C were reported in 54.6% and 42.4% of patients, respectively. No patients presented with Dukes stages A or D. HLA DRB3 was the most frequent allele detected followed by DRB4 and DR17. A higher frequency of the DRB3 allele was found in the peripheral blood when compared with the tumor and apparently normal tissues. HLA DRB3 and DR7 allele frequencies correlated with Duke's stages B and C but not with age, sex or degree of differentiation, while blood DR17 antigen correlated only with the degree of tumor differentiation.Conclusion: CRC was found to have a higher occurrence in younger patients. Tumors were aggressive with advance Duke's stage at presentation. This aggressive nature could possibly be related to either increased HLA DRB3 or DR7 or decreased HLA DR17 levels in the tumor tissue when compared with the blood. No differences between tumor and normal colon tissues were found, in concordance with the multifocally of colon cancer theory.

Expression of Lymphocyte Activation Gene 3 (LAG-3/CD223) by Chronic Lymphocytic Leukemia B Cells.

The clinical course of chronic lymphocytic leukemia (CLL) is variable. DNA-microarray studies have shown that the gene-expression patterns of CLL cells with unmutated IgVH genes are similar to those of cells with mutated IgVH genes, but that the patterns of both are distinct from those of other leukemias and lymphomas. Nevertheless, the two subtypes of CLL can be distinguished by the differential expression of a small number of genes, one of which encodes ZAP-70, an intracellular tyrosine kinase with a critical role in T-cell receptor signaling. Further analysis of the mutation status of IgVH genes and ZAP-70 expression revealed that CLL patients with B cells expressing ZAP-70 and unmutated IgVH genes had a more aggressive disease. LAG-3 (CD223) is thought to play a role in immune responses mediated by T and NK cells. LAG-3, a CD4 homolog, is a ligand for MHC class II antigens. Similar to ZAP-70, LAG-3 is selectively expressed on activated T and NK cells and has recently been shown to be expressed on T-cell activated B cells. We compared the gene expression profiles of the B cells purified from 15 CLL patients using the Affymetrix HG-U133 plus 2.0. This analysis revealed LAG-3 and parathymosin differentially expressed at higher levels by the CLL cells expressing ZAP-70 and unmutated IgVH genes. LAG-3 and parathymosin are located on chromosome 12p13 with head-to-tail orientation. To examine for surface expression of LAG-3, we performed flow cytometry on these same 15 CLL samples using an anti LAG-3 mAb (CD223). We found LAG-3 expressed at high levels by the CD5/CD19 B cells of 4/8 (50%) cases that expressed ZAP-70 and unmutated IgVH genes. Conversely, the samples with B cells lacking ZAP-70 and with mutated IgVH genes did not express LAG-3 (0/7). All samples (15/15) expressed high level of MHC class II antigens, as assessed by flow cytometry. LAG-3 may interact with MHC class II molecules expressed by CLL cells to form an autocrine loop that may further enhance the activation of ZAP-70-expressing CLL B cells. Further analysis is needed to delineate the role of LAG-3 in the pathogenesis and/or progression of this disease.