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HLA-B27-Homodimer-Specific Antibody Modulates the Expansion of Pro-Inflammatory T-Cells in HLA-B27 Transgenic Rats.

Marroquin Belaunzaran O, Kleber S, Schauer S, Hausmann M, Nicholls F, Van den Broek M, Payeli S, Ciurea A, Milling S, Stenner F, Shaw J, Kollnberger S, Bowness P, Petrausch U, Renner C - PLoS ONE (2015)

Bottom Line: HD5 bound B272 with high specificity and affinity (Kd = 0.32 nM).HD5 blocked cell-surface interaction of B272 with immune regulatory receptors KIR3DL2, LILRB2 and Pirb.In addition, HD5 modulated the production of TNF from CD4+ T-cells by limiting B272 interactions in vitro.

View Article: PubMed Central - PubMed

Affiliation: Division of Oncology, University Hospital Zurich, Zurich, Switzerland.

ABSTRACT

Objectives: HLA-B27 is a common genetic risk factor for the development of Spondyloarthritides (SpA). HLA-B27 can misfold to form cell-surface heavy chain homodimers (B272) and induce pro-inflammatory responses that may lead to SpA pathogenesis. The presence of B272 can be detected on leukocytes of HLA-B27+ Ankylosing spondylitis (AS) patients and HLA-B27 transgenic rats. We characterized a novel B272-specific monoclonal antibody to study its therapeutic use in HLA-B27 associated disorders.

Methods: The monoclonal HD5 antibody was selected from a phage library to target cell-surface B272 homodimers and characterized for affinity, specificity and ligand binding. The immune modulating effect of HD5 was tested in HLA-B27 transgenic rats. Onset and progression of disease profiles were monitored during therapy. Cell-surface B272 and expansion of pro-inflammatory cells from blood, spleen and draining lymph nodes were assessed by flow cytometry.

Results: HD5 bound B272 with high specificity and affinity (Kd = 0.32 nM). HD5 blocked cell-surface interaction of B272 with immune regulatory receptors KIR3DL2, LILRB2 and Pirb. In addition, HD5 modulated the production of TNF from CD4+ T-cells by limiting B272 interactions in vitro. In an HLA-B27 transgenic rat model repetitive dosing of HD5 reduced the expansion of pro-inflammatory CD4+ T-cells, and decreased the levels of soluble TNF and number of cell-surface B272 molecules.

Conclusion: HD5 predominantly inhibits early TNF production and expansion of pro-inflammatory CD4+ T-cells in HLA-B27 transgenic rats. Monoclonal antibodies targeting cell-surface B272 propose a new concept for the modulation of inflammatory responses in HLA-B27 related disorders.

No MeSH data available.


Related in: MedlinePlus

Detection of cell-surface B272 in leukocyte populations of HLA-B27 transgenic rats at different ages.(A) Representative flow cytometry analysis of cell-surface B272 expression in leukocytes populations of Tg rats at different ages (6, 15, 23 and 30 weeks) from spleen and MLNs. WT leukocytes represent the control population where B272 is absent. (B) MFI values plotted of positive B272 stains from splenocytes. (C) MFI values plotted of positive B272 stains from MLNs. Detection of cell-surface B272 homodimers was performed using HD6-biotinylated and detected by streptavidin-APC. HD6 had been previously assessed as an antibody capable of recognizing cell-surface B272 in human [12] and rat [41] leukocyte populations. Antibody panels: CD4+ T-cells (+CD3, +CD4), CD8+ T-cells (+CD3, +CD8), NK (+CD161a,—lineage), B cells (+CD45RA,-lineage) and Monocytes (+CD172a,—RP-1).
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pone.0130811.g004: Detection of cell-surface B272 in leukocyte populations of HLA-B27 transgenic rats at different ages.(A) Representative flow cytometry analysis of cell-surface B272 expression in leukocytes populations of Tg rats at different ages (6, 15, 23 and 30 weeks) from spleen and MLNs. WT leukocytes represent the control population where B272 is absent. (B) MFI values plotted of positive B272 stains from splenocytes. (C) MFI values plotted of positive B272 stains from MLNs. Detection of cell-surface B272 homodimers was performed using HD6-biotinylated and detected by streptavidin-APC. HD6 had been previously assessed as an antibody capable of recognizing cell-surface B272 in human [12] and rat [41] leukocyte populations. Antibody panels: CD4+ T-cells (+CD3, +CD4), CD8+ T-cells (+CD3, +CD8), NK (+CD161a,—lineage), B cells (+CD45RA,-lineage) and Monocytes (+CD172a,—RP-1).

Mentions: We next examined the presence of cell-surface B272 in leukocyte populations in Tg rats at 6, 15, 23, and 30 weeks of age, to determine the different time points of B272 expression and relation to disease progression. Results showed positive staining for B272 in all leukocyte populations examined except granulocytes (Fig 4A). Cell-surface B272 was almost absent in the spleen of young rats (6 weeks), but increased with age reaching a maximum between 15 and 23 weeks (Fig 4A and 4B). Lymphocytes from the draining lymph nodes of the gut (mesenchymal lymph nodes (MLN)) of Tg rats where inflammatory processes are associated to colitis displayed higher numbers of B272 dimers as early as 6 weeks, and increased with age at higher rates when compared to splenocytes (Fig 4A–4C).


HLA-B27-Homodimer-Specific Antibody Modulates the Expansion of Pro-Inflammatory T-Cells in HLA-B27 Transgenic Rats.

Marroquin Belaunzaran O, Kleber S, Schauer S, Hausmann M, Nicholls F, Van den Broek M, Payeli S, Ciurea A, Milling S, Stenner F, Shaw J, Kollnberger S, Bowness P, Petrausch U, Renner C - PLoS ONE (2015)

Detection of cell-surface B272 in leukocyte populations of HLA-B27 transgenic rats at different ages.(A) Representative flow cytometry analysis of cell-surface B272 expression in leukocytes populations of Tg rats at different ages (6, 15, 23 and 30 weeks) from spleen and MLNs. WT leukocytes represent the control population where B272 is absent. (B) MFI values plotted of positive B272 stains from splenocytes. (C) MFI values plotted of positive B272 stains from MLNs. Detection of cell-surface B272 homodimers was performed using HD6-biotinylated and detected by streptavidin-APC. HD6 had been previously assessed as an antibody capable of recognizing cell-surface B272 in human [12] and rat [41] leukocyte populations. Antibody panels: CD4+ T-cells (+CD3, +CD4), CD8+ T-cells (+CD3, +CD8), NK (+CD161a,—lineage), B cells (+CD45RA,-lineage) and Monocytes (+CD172a,—RP-1).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4488392&req=5

pone.0130811.g004: Detection of cell-surface B272 in leukocyte populations of HLA-B27 transgenic rats at different ages.(A) Representative flow cytometry analysis of cell-surface B272 expression in leukocytes populations of Tg rats at different ages (6, 15, 23 and 30 weeks) from spleen and MLNs. WT leukocytes represent the control population where B272 is absent. (B) MFI values plotted of positive B272 stains from splenocytes. (C) MFI values plotted of positive B272 stains from MLNs. Detection of cell-surface B272 homodimers was performed using HD6-biotinylated and detected by streptavidin-APC. HD6 had been previously assessed as an antibody capable of recognizing cell-surface B272 in human [12] and rat [41] leukocyte populations. Antibody panels: CD4+ T-cells (+CD3, +CD4), CD8+ T-cells (+CD3, +CD8), NK (+CD161a,—lineage), B cells (+CD45RA,-lineage) and Monocytes (+CD172a,—RP-1).
Mentions: We next examined the presence of cell-surface B272 in leukocyte populations in Tg rats at 6, 15, 23, and 30 weeks of age, to determine the different time points of B272 expression and relation to disease progression. Results showed positive staining for B272 in all leukocyte populations examined except granulocytes (Fig 4A). Cell-surface B272 was almost absent in the spleen of young rats (6 weeks), but increased with age reaching a maximum between 15 and 23 weeks (Fig 4A and 4B). Lymphocytes from the draining lymph nodes of the gut (mesenchymal lymph nodes (MLN)) of Tg rats where inflammatory processes are associated to colitis displayed higher numbers of B272 dimers as early as 6 weeks, and increased with age at higher rates when compared to splenocytes (Fig 4A–4C).

Bottom Line: HD5 bound B272 with high specificity and affinity (Kd = 0.32 nM).HD5 blocked cell-surface interaction of B272 with immune regulatory receptors KIR3DL2, LILRB2 and Pirb.In addition, HD5 modulated the production of TNF from CD4+ T-cells by limiting B272 interactions in vitro.

View Article: PubMed Central - PubMed

Affiliation: Division of Oncology, University Hospital Zurich, Zurich, Switzerland.

ABSTRACT

Objectives: HLA-B27 is a common genetic risk factor for the development of Spondyloarthritides (SpA). HLA-B27 can misfold to form cell-surface heavy chain homodimers (B272) and induce pro-inflammatory responses that may lead to SpA pathogenesis. The presence of B272 can be detected on leukocytes of HLA-B27+ Ankylosing spondylitis (AS) patients and HLA-B27 transgenic rats. We characterized a novel B272-specific monoclonal antibody to study its therapeutic use in HLA-B27 associated disorders.

Methods: The monoclonal HD5 antibody was selected from a phage library to target cell-surface B272 homodimers and characterized for affinity, specificity and ligand binding. The immune modulating effect of HD5 was tested in HLA-B27 transgenic rats. Onset and progression of disease profiles were monitored during therapy. Cell-surface B272 and expansion of pro-inflammatory cells from blood, spleen and draining lymph nodes were assessed by flow cytometry.

Results: HD5 bound B272 with high specificity and affinity (Kd = 0.32 nM). HD5 blocked cell-surface interaction of B272 with immune regulatory receptors KIR3DL2, LILRB2 and Pirb. In addition, HD5 modulated the production of TNF from CD4+ T-cells by limiting B272 interactions in vitro. In an HLA-B27 transgenic rat model repetitive dosing of HD5 reduced the expansion of pro-inflammatory CD4+ T-cells, and decreased the levels of soluble TNF and number of cell-surface B272 molecules.

Conclusion: HD5 predominantly inhibits early TNF production and expansion of pro-inflammatory CD4+ T-cells in HLA-B27 transgenic rats. Monoclonal antibodies targeting cell-surface B272 propose a new concept for the modulation of inflammatory responses in HLA-B27 related disorders.

No MeSH data available.


Related in: MedlinePlus