Derek Hart

cDNA cloning of human DEC-205, a putative antigen-uptake receptor on dendritic cells.

Abstract Dendritic cells (DC) are specialist antigen presenting cells which capture antigens in the periphery, migrate centrally, and present the processed antigens in the context of major histocompatibility complex and appropriate co-stimulatory molecules to T lymphocytes for the initiation of an immune response. DEC-205 has been identified as a putative antigen-uptake receptor, which is expressed abundantly on mouse DC. The recently cloned mouse DEC-205 cDNA predicts a molecular structure which has a marked similarity to the macrophage mannose receptor. Using reverse transcriptase-polymerase chain reaction (RT-PCR) and cDNA library screening, we obtained the full coding region of human DEC-205 cDNA from the Hodgkin’s disease-derived L428 cell line. The predicted protein structure is a type I transmembrane protein of 1722 amino acids consisting of a signal peptide, cysteine-rich domain, fibronectin type II domain, ten carbohydrate recognition-like domains, transmembrane domain, and a cytoplasmic tail. Human DEC-205 is 77% identical to the mouse protein with completely conserved cysteines. The DEC-205 gene (LY75) was mapped to chromosome band 2q24 by somatic cell hybrid panel analysis and fluorescent in situ hybridization. Northern blot analysis detected 7.8 and 9.5 kilobase DEC-205 transcripts in myeloid, B lymphoid, and Hodgkin’s disease-derived cell lines. RT-PCR analysis indicated that immature blood DC contain a barely detectable amount of DEC-205 transcripts but these were markedly increased upon differentiation/activation.

Expression of the RelB transcription factor correlates with the activation of human dendritic cells.

The RelB gene product is a member of the nuclear factor (NF)‐κB family of transcription factors. It has been identified recently within mouse antigen‐presenting cells and human monocyte‐derived dendritic cells (DC). Disruption of the mouse RelB gene is accompanied, amongst other phenotypes, by abnormalities in the antigen‐presenting cell lineages. In order to define RelB expression during human DC differentiation, we have analysed RelB mRNA by reverse transcriptase–polymerase chain reaction and RelB protein by intracellular staining in CD34+ precursors and different types of DC preparations. RelB mRNA was not detected in CD34+ precursor populations. Fresh blood DC (lineage−human leucocyte antigen‐DR+ (lin−HLA‐DR+)) lacked RelB mRNA and cytoplasmic RelB protein but a period of in vitro culture induced RelB expression in blood DC. Purified Langerhans’ cells (LC) (CD1a+ HLA‐DR+) failed to express RelB mRNA. Immunocytochemical staining identified RelB protein in human skin epithelium. RelB protein was expressed in a very few CD1a+, CD83+ or CMRF‐44+ dermal DC but was not present in CD1a+ LC. Tonsil DC (lin−HLA‐DR+ CMRF‐44+) were positive for RelB mRNA and RelB protein. Intestinal DC (HLA‐DR+) also lacked immunoreactive RelB protein. The majority of interdigitating CD83+, CMRF‐44+, CMRF‐56+ or p55+ DC located in paracortical T‐lymphocyte areas of lymph node and tonsil contained RelB protein. The expression of RelB mRNA and RelB protein correlates with the activated phase of blood DC and the postmigration cell (activated) stage of tissue DC development.

CHAPTER 8 – Phenotypic characterization of dendritic cells

The phenotype of dendritic cells (DCs) migrating through the tissues prior to interaction with T lymphocytes relates to adhesion molecule and chemokine receptor expression. DCs express a wide variety of adhesion molecules. The ligands for CD11a (LFA-1), CD54 (ICAM-1), CD50 (ICAM-2), and CD102 (ICAM-3) are all expressed on DCs but show differential regulation. CD54 is expressed at low density on blood DCs and Langerhans cells (LCs) but is quickly upregulated by activation. E-cadherin has a role in migration and is expressed by mouse and human LCs, and blood DCs but downregulates from LCs as the cells migrate. Interaction between DCs and the connective tissue may be stabilized by isoforms of the CD44 molecule that is expressed in high density by DCs. The DCIR molecule, which is structurally related to the C type lectins, contains a cytoplasmic inhibitory motif and is expressed by lin – DCs, Mo-DCs, and CD34 + -derived DCs of the myeloid phenotype and not those with a LC phenotype.

Reference: CD Antigens 2002.

The Proceedings of the 7th Human Leukocyte Differentiation Antigen (HLDA) Workshop are about to be published, detailing more than 80 new CD specificities. The next Workshop, planned for 2004, will continue this process, and a number of candidate CD molecules in the literature, identified by antibody

Characterization of the New Immune Suppressive Anti Human CD83 Monoclonal Antibody 3C12C in Non-Human Primates

Introduction CD83 is a member of the Ig superfamily. It is membrane bound on activated dendritic cells (DC) and B cells. We developed a potential therapeutic human anti-human CD83 monoclonal antibody (3C12C) and showed it prevents graft versus host disease in a human PBMC mouse xenograft model. After establishing that 3C12C binds to non-human primate (NHP, baboon Papio Hamadryas) cells, we tested 3C12C in vivo in NHPs before a first in man, first in class, Phase I clinical trial. Methods Five baboons received 3C12C (1, 5, 10mg/kg, iv) or human IgG 10mg/kg on d0, 7, 14 and d21. Peripheral blood and serum were collected weekly (x4) then every 4 weeks (x2). Bone marrow and lymph node (LN) biopsies were taken at d28. Blood counts and biochemistry were monitored. Flow cytometry analysis followed the PBMC DC subsets, B cells and T cells and bone marrow haematopoietic stem cells. Immune histological studies were performed on LNs. Results All 5 animals remained well following anti-CD83 antibody injection. 3C12C did not change blood counts or liver function. CD4+T, CD8+T and B cells remained normal to d84. 3C12C injection increased peripheral Treg transiently at d21. 3C12C reduced blood CD1c+DC in a dose dependent manner. CD1c+DC were reduced in LN. 3C12C had no influence on bone marrow hematopoietic stem cell numbers. Conclusion Our results demonstrate that 3C12C is safe in NHPs and that it reduced activated DC numbers. This data will facilitate our planned Phase I trial of 3C12C in allogenic hematopoietic stem cell transplantation.