Ian C. Nicholson

Construction and characterisation of a functional CD19 specific single chain Fv fragment for immunotherapy of B lineage leukaemia and lymphoma

The B cell specific antigen CD19 is a target for the immunotherapy of B lineage leukaemias and lymphomas. We have engineered a single chain Fv (scFv) fragment from the mouse hybridoma cell line FMC63 which produces monoclonal antibody specific for CD19. The genes encoding the FMC63 heavy and light chain variable regions were amplified from cDNA and a scFv was constructed by splice overlap extension PCR. Analysis of staining of lymphoblastoid cell lines, peripheral blood lymphocytes and tonsil sections demonstrated that the monovalent scFv fragment has the same cellular specificity as the parent hybridoma antibody. Kinetic studies with radiolabelled material showed that the scFv binds target cells with a Ka of 2.3 x 10(-9), compared with 4.2 x 10(-9) for the parent antibody. This CD19 scFv will be used in experimental models to test its therapeutic efficacy and immunogenicity, with a view to application in the diagnosis and treatment of human B cell cancers.

ProNGF mediates death of Natural Killer cells through activation of the p75NTR–sortilin complex

The common neurotrophin receptor P75NTR, its co-receptor sortilin and ligand proNGF, have not previously been investigated in Natural Killer (NK) cell function. We found freshly isolated NK cells express sortilin but not significant amounts of P75NTR unless exposed to interleukin-12 (IL-12), or cultured in serum free conditions, suggesting this receptor is sequestered. A second messenger associated with p75NTR, neurotrophin-receptor-interacting-MAGE-homologue (NRAGE) was identified in NK cells. Cleavage resistant proNGF123 killed NK cells in the presence of IL-12 after 20h and without IL-12 in serum free conditions at 48h. This was reduced by blocking sortilin with neurotensin. We conclude that proNGF induced apoptosis of NK cells may have important implications for limiting the innate immune response.

Expression of Toll-like receptors by neonatal leukocytes.

To cite this article: Dasari P, Zola H, Nicholson IC. Expression of Toll‐like receptors by neonatal leukocytes. Pediatr Allergy Immunol 2011; 22: 221–228.

Characterization of a Distinct Population of Circulating Human Non-Adherent Endothelial Forming Cells and Their Recruitment via Intercellular Adhesion Molecule-3

Circulating vascular progenitor cells contribute to the pathological vasculogenesis of cancer whilst on the other hand offer much promise in therapeutic revascularization in post-occlusion intervention in cardiovascular disease. However, their characterization has been hampered by the many variables to produce them as well as their described phenotypic and functional heterogeneity. Herein we have isolated, enriched for and then characterized a human umbilical cord blood derived CD133+ population of non-adherent endothelial forming cells (naEFCs) which expressed the hematopoietic progenitor cell markers (CD133, CD34, CD117, CD90 and CD38) together with mature endothelial cell markers (VEGFR2, CD144 and CD31). These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8) or myeloid markers (CD11b and CD14) which distinguishes them from ‘early’ endothelial progenitor cells (EPCs). Functional studies demonstrated that these naEFCs (i) bound Ulex europaeus lectin, (ii) demonstrated acetylated-low density lipoprotein uptake, (iii) increased vascular cell adhesion molecule (VCAM-1) surface expression in response to tumor necrosis factor and (iv) in co-culture with mature endothelial cells increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human endothelial cells (ECs). Extensive genomic and proteomic analyses of the naEFCs showed that intercellular adhesion molecule (ICAM)-3 is expressed on their cell surface but not on mature endothelial cells. Furthermore, functional analysis demonstrated that ICAM-3 mediated the rolling and adhesive events of the naEFCs under shear stress. We suggest that the distinct population of naEFCs identified and characterized here represents a new valuable therapeutic target to control aberrant vasculogenesis.

Monoclonal antibodies to human cell surface antigens.

Many of the leukocyte cell surface molecules are known by “CD” numbers. In this Appendix, a short introduction describes the history and the use of CD nomenclature and provides a few key references to enable access to the wider literature. This is followed by a table that lists all human molecules with approved CD names, tabulating alternative names, key structural features, cellular expression, major known functions, and usefulness of the molecules or antibodies against them in research or clinical applications. Curr. Protoc. Immunol. 80:A.4A.1‐A.4A.73.

Antibody against CD20 in patients with B cell malignancy

Cancer patients may make antibodies against antigens on the surface of their malignant cells due either to the expression of unique antigens or to dysregulated responses to self antigens. Patients with B cell malignancy frequently produce autoantibodies and may therefore be a source of immunoglobulin genes for the production of phage display antibody libraries directed against tumour-associated antigens. Patients with autoimmune disease have circulating antibodies against lymphocyte surface antigens, and may also provide a good starting point for the production of a library of lymphocyte-reactive antibody structures. In this study, plasma and serum samples from patients with B cell malignancy or Sjogrens syndrome and from healthy controls were screened for antibodies against the B cell membrane antigens CD20. While the majority of samples showed very low reactivity, some individuals did show significant and reproducible binding to CD20. To identify a good donor for library construction, it would be advisable to screen donors for antibody against the antigens of interest.

PI16 is expressed by a subset of human memory Treg with enhanced migration to CCL17 and CCL20.

The peptidase inhibitor PI16 was shown previously by microarray analysis to be over-expressed by CD4-positive/CD25-positive Treg compared with CD4-positive/CD25-negative Th cells. Using a monoclonal antibody to the human PI16 protein, we found that PI16-positive Treg have a memory (CD45RO-positive) phenotype and express higher levels of FOXP3 than PI16-negative Treg. PI16-positive Treg are functional in suppressor assays in vitro with potency similar to PI16-negative Treg. Further phenotyping of the PI16-positive Treg revealed that the chemokine receptors CCR4 and CCR6 are expressed by more of the PI16-positive/CD45RO-positive Treg compared with PI16-negative/CD45RO-positive Treg or Th cells. PI16-positive Treg showed enhanced in vitro migration towards the inflammatory chemokines CCL17 and CCL20, suggesting they can migrate to sites of inflammation. We conclude that PI16 identifies a novel distinct subset of functional memory Treg which can migrate to sites of inflammation and regulate the pro-inflammatory response at those sites.

Tolerization as a tool for generating novel monoclonal antibodies

Standard hybridoma production involves the fusion of spleen cells from an immunized mouse with a non‐secretory murine myeloma cell line. While this technology has provided numerous reagents that are highly valuable, demand is now increasing for monoclonal antibodies which can distinguish between closely related antigens. Induction of tolerance towards common antigens enables the recovery of high‐specificity reagents that have previously proved elusive. This review details a number of strategies using either complex protein mixtures or purified proteins as tolerogens and subsequent immunization with a closely related immunogen.

Comparison of blood and synovial fluid Th17 and novel peptidase inhibitor 16 Treg cell subsets in juvenile idiopathic arthritis

Objective. Early recognition and treatment of juvenile idiopathic arthritis (JIA) can prevent joint damage and minimize side effects of medication. The balance between proinflammatory and antiinflammatory mechanisms is known to be important in JIA, and we therefore investigated T cell subsets including Th cells, autoaggressive Th17 cells, and regulatory T cells (Treg), including a novel Treg subset in peripheral blood (PB) and synovial fluid (SF) of patients with JIA. Methods. Fifty children with JIA were enrolled in our study. Frequency, phenotype, and function of T lymphocytes in PB and SF were characterized using flow cytometry. Migration capabilities of PB and SF cells were compared. Results. Synovial T cells showed different phenotype and function compared with PB T cells, with an increased proportion of memory T cells, expression of CCR4, CCR5, CXCR3, interleukin 23R, and an increased ratio of Th17 to Treg. Although Treg were increased in SF compared with the PB, we found a significant decrease in the numbers of peptidase inhibitor 16 (PI16)+ Treg in active joints compared with peripheral blood. Coexpression of CCR4 and CCR6 was reduced on PI16+ Treg in PB and SF of patients with JIA compared with healthy children, however the ability of these cells to migrate toward their ligands was unaffected. Conclusion. This is a comprehensive characterization of novel PI16+ Treg and Th17 cells in matched blood and synovial fluid samples of patients with JIA. Despite an increased number of Treg within the inflamed joint, lower numbers of PI16+ Treg but high numbers of Th17 cells might contribute to the inability to control disease.

Rapid detection of immunoglobulin gene somatic hypermutation using heteroduplex formation

In order to identify somatic hypermutation of rearranged human immunoglobulin genes, we have examined heteroduplex formation between cloned VH6 genes. In test systems, the presence of five or more point mutations could be detected by examining the formation of heteroduplexes between a known germline VH6 gene and other sequenced genes using polyacrylamide gel electrophoresis. If a mutated sequence was used, then the presence of two or more mutations could be detected. The method was used for rapid screening of VH6-D-JH rearrangements for the presence of point mutations before sequencing, and to distinguish between different highly mutated sequences, allowing clones containing the same rearrangement to be identified indirectly.