Surface LSP-1 Is a Phenotypic Marker Distinguishing Human Classical versus Monocyte-Derived Dendritic Cells.

Human mononuclear phagocytes comprise several subsets of dendritic cells (DCs), monocytes, and macrophages. Distinguishing one population from another is challenging, especially in inflamed tissues, owing to the promiscuous expression of phenotypic markers. Using a synthetic library of humanized llama single domain antibodies, we identified a novel surface marker for human naturally occurring monocyte-derived DCs. Our antibody targets an extra-cellular domain of LSP-1, specifically on monocyte-derived DCs, but not on other leukocytes, in particular monocytes, macrophages, classical DCs, or the recently described blood DC3 population. Our findings will pave the way for a better characterization of human mononuclear phagocytes in pathological settings.

Bacterial cytoplasm as an effective cell compartment for producing functional VHH-based affinity reagents and Camelidae IgG-like recombinant antibodies.

BACKGROUND: The isolation of recombinant antibody fragments from displayed libraries represents a powerful alternative to the generation of IgGs using hybridoma technology. The selected antibody fragments can then be easily engineered into (multi)-tagged constructs of variable mass and complexity as well as reconstituted into Camelidae IgG-like molecules when expressed fused to Fc domains. Nevertheless, all antibody constructs depend on an oxidizing environment for correct folding and consequently still belong to the proteins difficult to express in bacteria. In such organisms they are mostly produced at low yields in the periplasmic space. RESULTS: We demonstrate that fusion constructs of recombinant antibodies in combination with multiple tags can be produced at high yields and totally functional in the cytoplasm of bacteria expressing sulfhydryl oxidase. The method was applied to structurally demanding molecules such as VHHs fused to SNAP and Fc domains and was validated using the antibody-derived reagents in a variety of immune techniques (FACS, ELISA, WB, IP, SPR, and IF). CONCLUSIONS: The collected data demonstrate the feasibility of a method that establishes a totally new approach for producing rapidly and inexpensively functional Camelidae IgG-like monoclonal antibodies and antibody-based reagents containing multiple disulfide bonds and suitable for both basic research and clinical applications.

Hierarchy of CD4 T cell epitopes of the ANRS Lipo5 synthetic vaccine relies on the frequencies of pre-existing peptide-specific T cells in healthy donors.

The Agence National de Recherche sur le SIDA et les hepatitis Lipo5 vaccine is composed by five long fragments of HIV proteins and was recently shown to induce in seronegative volunteers a CD4 T cell response largely dominated by the G2 fragment. To understand this response profile, we submitted the five HIV fragments to HLA-DR-binding assays and evaluated the frequency of naive Lipo5-specific CD4 T lymphocytes in the blood of 22 healthy individuals. We enumerated the Lipo5-specific T cell lines induced in vitro by weekly rounds of specific stimulation. Four peptides and hence not only G2 exhibited a broad specificity for HLA-DR molecules. In contrast, most of the T cell lines specific for Lipo5 reacted with G2, revealing a G2-specific T cell repertoire superior to 2 cells per million, whereas it is close to 0.4 for the other peptides. We also found good cross-reactivity of all the peptides with clade B and C variants and that G2 and P1 are able to recruit T cells that recognize HIV-infected cells. We therefore mainly observed very good concordance between the frequency to individual Lipo5 peptides among vaccinees in a large-scale vaccine trial and the distribution of peptide specificity of the in vitro induced T cell lines. These findings underline the role of the size of the epitope-specific naive repertoire in shaping the CD4 T cell response after vaccination and highlight the value of evaluating the naive repertoire to predict vaccine immunogenicity.

The signal peptide of the tumor-shared antigen midkine hosts CD4+ T cell epitopes.

BACKGROUND: The CD4 T cell response to the tumor antigen Midkine was unknown. RESULTS: Most of the T cell response to Midkine relies on T cell epitopes contained in its signal peptide. CONCLUSION: The signal peptide of Midkine is accessible to HLA class II pathway for CD4 T cell presentation. SIGNIFICANCE: It is a new function for signal peptides to contribute to tumor-specific CD4 T cell response. Because of the key role of CD4 T cell response in immunity to tumors, we investigated the CD4(+) T cell response to the recently identified tumor antigen Midkine (MDK). By weekly stimulations of T lymphocytes harvested from seven HLA-DR-typed healthy donors, we derived CD4(+) T cell lines specific for eight MDK peptides. Most of the T cell lines reacted with the peptides 9-23 and 14-28, located in and overlapping the MDK signal peptide, respectively. Accordingly, the MDK signal peptide appeared to be rich in good binders to common HLA-DR molecules. The peptide 9-23-specific T cell lines were specifically stimulated by autologous dendritic cells loaded with lysates of MDK-transfected cells or with lysates of tumor cells naturally expressing the MDK protein. One T cell line was stimulated by HLA-compatible MDK-transfected tumor cells. By contrast, the peptide 14-28-specific T cell lines were not stimulated in any of these conditions. Our data demonstrate that CD4(+) T cell epitopes present in the signal peptide can be accessible to recognition by CD4(+) T cells and may therefore contribute to tumor immunity, whereas a peptide overlapping the junction between the signal peptide and the mature protein is not.